Delivery of a light-activated cation channel into the brain of a subject

ABSTRACT

The present invention provides compositions and methods for light-activated cation channel proteins and their uses within cell membranes and subcellular regions. The invention provides for proteins, nucleic acids, vectors and methods for genetically targeted expression of light-activated cation channels to specific cells or defined cell populations. In particular the invention provides millisecond-timescale temporal control of cation channels using moderate light intensities in cells, cell lines, transgenic animals, and humans. The invention provides for optically generating electrical spikes in nerve cells and other excitable cells useful for driving neuronal networks, drug screening, and therapy.

CROSS-REFERENCE

This application is a divisional under 35 U.S.C. § 120 of U.S. patentapplication Ser. No. 11/459,637 filed on Jul. 24, 2006; which claimsbenefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No.60/701,799 filed Jul. 22, 2005, which are fully incorporated herein byreference in their entirety.

FEDERALLY-SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under contracts DC007006and MH071315 awarded by the National Institutes of Health. TheGovernment has certain rights in the invention.

FIELD OF THE INVENTION

The invention relates to light-activated ion channel proteins that cangenerate millisecond-timescale electrical spikes when incorporated intoneurons and illuminated with rapid pulses of light. In particular theinvention provides millisecond-timescale temporal control of cationchannels using moderate light intensities in cells, cell lines,transgenic animals, and humans. The invention is useful for drivingneuronal networks, for drug screening, and for therapy.

BACKGROUND OF THE INVENTION

Ion channel proteins control the flow of ions across membranes, forinstance, between the cytoplasm and the outside of a cell. A cationchannel operates by controlling the flow of cations such as sodium,potassium, calcium, lithium, rubidium, and cesium. When the cationchannel is closed, the transport of cations across the membranes isslow, when the cation channel opens, the flow of cations through thechannel increases. If the opening of the channel results in a net flowof cations to one side of the membrane, an electrical current will begenerated. The flow of ions across the membrane can also result in achange in the voltage across the membrane. If there is a net voltageacross the membrane at the time the channel is opened, cations will tendto flow so as to cause depolarization of the membrane. Neurons (nervecells) use rapid depolarizations to create action potentials (spikes)creating electrical signals that propagate down the neuron. These actionpotentials, nerve impulses, or spikes occur on the millisecond timescale, and they are the basis by which the neuron acts to signal, andcontrol brain and muscle function.

Neurons receive, conduct, and transmit signals. In a motor neuron, thesignals represent commands for the contraction of a particular muscle.In a sensory neuron, signals represent the information that a specifictype of stimulus is present. In an interneuron, signals represent partof a computation that combines sensory information from many differentsources and generates an appropriate set of motor commands in response.Communication depends on an electrical disturbance in one part of themembrane spreading to other parts of the cell. These communications areoften via an action potential, also referred to as a spike or a nerveimpulse. Neuronal signals are transmitted from cell to cell at synapseswhich are specialized sites of cell contact. Synapses can be eitherelectrical synapses (gap junctions), or chemical synapses. The usualmechanism of communication across a chemical synapse involves a changein electrical potential within a first (presynaptic) neuron that resultsin the release of neurotransmitter. The neurotransmitter diffuses to asecond (postsynaptic) neuron across the gap between the neurons (thesynaptic cleft). The neurotransmitter can provoke an electrical responsein the postsynaptic neuron. The change created at the synapse due to theelectrical signal from the presynaptic neuron is a synaptic event.Synaptic events can be excitatory or inhibitory. By controlling synapticevents, the control of transmission of signals between neurons can becontrolled, allowing optical driving of activity throughout a connectedneural network.

Noninvasive temporal control of activity in defined neuronal populationsis a long-sought goal of neuroscience. In the mammalian nervous system,it is believed that neural computation depends on the temporallydiverse, precise spiking patterns of different classes of neurons, whichexpress unique genetic markers and display heterogeneous morphologicaland wiring properties (e.g. Pouille et al., Nature 429:717 (2004),Nirenberg et al., Neuron 18:637 (1997), Klausberger et al., Nature421:844 (2003), Hausser et al., Neuron 19:665 (1997)) within connectednetworks. While direct field stimulation and recording of neurons inintact brain tissue have provided many insights into the causal functionof circuit subfields (Kandel et al., J Neurophysiol 24:243 (1961),Kandel et al., J Neurophysiol 24:225 (1961), Ditterich et al., NatNeurosci 6:891 (2003), Gold et al., Nature 404:390 (2000), Salzman etal., Nature 346:174 (1990), neurons belonging to a specific class areoften sparsely embedded within dense tissue, posing fundamentalchallenges for resolving the causal role of particular neuron types ininformation processing.

For many cellular and systems neuroscience processes (and for nonspikingneurons in species like C. elegans), subthreshold depolarizations conveyinformation of physiological significance. For example, subthresholddepolarizations are highly potent for activating synapse-to-nucleussignaling (Mermelstein et al., J. Neurosci. 20:266 (2000)), and therelative timing of subthreshold and suprathreshold depolarizations iscritical for determining the sign of synaptic plasticity (Bi et al., J.Neurosci. 18:10464 (1998)). But compared with driving spiking, it is inprinciple a more difficult task to drive reliable and precisely sizedsubthreshold depolarizations. The sharp threshold for action potentialproduction facilitates reliable spiking, while the all-or-none dynamicsof spiking produces virtually identical waveforms from spike to spike,even in the presence of significant neuron-to-neuron variability inelectrical properties. In contrast, subthreshold depolarizations, whichoperate in the linear regime of membrane voltage, will lack theseintrinsic normalizing mechanisms.

Despite the progress made in the analysis of neural network geometry vianon-cell type specific techniques like glutamate uncaging (e.g. Shepherdet al., Neuron 38:277 (2003), Denk et al., J Neurosci Methods 54:151(1994), Pettit et al., J Neurophysiol 81:1424 (1999), Yoshimura et al.,Nature 433:868 (2005), Dalva et al., Science 265:255 (1994), Katz etal., J Neurosci Methods 54:205 (1994)), no non-invasive technology hasyet been invented with the requisite spatiotemporal resolution to probeneural coding in specific neurons at the resolution of single spikes.Previously genetically-encoded optical methods, have demonstratedcontrol of neuronal function only over timescales of seconds to minutes,perhaps due to the nature of their membrane potential controlmechanisms. Kinetics roughly a thousand times faster would enable remotecontrol of individual spikes or synaptic events. Thus, existinggenetically-targeted approaches require expensive, custom-synthesizedexogenous compounds, and operate on the scale of seconds to minutes(Lima et al., Cell 121:141 (2005), Banghart et al., Nat Neurosci 7:1381(2004), Zemelman et al., Proc Natl Acad Sci USA 100:1352 (2003), Fosteret al., Nature 433:698 (2005)).

Thus, compositions and methods for higher-temporal resolution,noninvasive, and genetically-based control of neural activity would bedesirable.

SUMMARY OF THE INVENTION

Accordingly, one aspect of the invention provides for compositions andmethods related to light-activated cation channel (LACC) proteins withmillisecond-scale opening kinetics that provide genetically targetedphotostimulation at fine temporal resolution, enabling elucidation ofthe temporal activity patterns in specific neurons that suffice to drivecircuit dynamics, information processing, and plasticity. Thecomposition and methods herein offer optical control of the electricaland ionic milieu of neurons and a variety of other cell types, both invitro and in vivo, at the rapid timescales important for many biologicalprocesses—including ion channel modulation, signal transduction, neuralcoding via temporal spiking and synaptic activation patterns, sensoryand motor processing, interneuron modulation of circuit dynamics, andneuropsychiatric dysfunction. Additional aspects of the inventioninclude compositions and methods relating to light-activated cationchannel proteins for drug discovery and biotechnological andneuropsychiatric applications, advancing the ability to characterize,detect, and treat a variety of medical disorders.

Another aspect of the present invention is a composition comprising alight-activated cation channel protein that is expressed in a cellwherein the cell is selected from the group consisting of mammaliancells, neurons, and stem cells. In one embodiment, the light-activatedcation channel protein is ChR2, Chop2, ChR2-310, or Chop2-310. Inanother embodiment, the light-activated cation channel protein is a7-transmembrane protein. In another embodiment, the light-activatedcation channel protein is a single-component protein. In yet anotherembodiment, the LACC protein covalently binds retinal.

Still another aspect of the present invention is an isolated LACCprotein that responds to a light stimulus within 1 ms.

Another aspect of the present invention is an isolated LACC protein thatgenerates a stable photocurrent that does not increase or decrease byabout 10% over about 60 min.

Another aspect of the present invention is an isolated LACC protein thatgenerates electrical responses with a temporal jitter of less than about5 ms.

Another aspect of the present invention is an isolated LACC protein thatcan generate subthreshold pulses with a coefficient of variation of lessthan about 0.2 over about 5 pulses.

Another aspect of the present invention is a nucleic acid sequencecomprising a gene for LACC protein and a promoter. In one embodiment,the promoter is a cell specific promoter. In another embodiment, thepromoter is a promoter for somatostatin, parvalbumin, GABAα6, L7, orcalbindin. In another embodiment, the promoter is a cell general purposepromoter. In another embodiment, the promoter is EF1-alpha. In anotherembodiment, the nucleic acid sequence comprises a bacterial artificialchromosome (BAC). In another preferred embodiment, the promoter is aninducible promoter, such as a promoter inducible by a trans-actingfactor which can respond to an administered drug.

Another aspect of the present invention is a fusion protein comprising aLACC protein coupled to another functional protein. In one embodiment,the other functional protein is a fluorescent protein. In oneembodiment, the other functional protein is mCherry, GFP, YFP, or CFP.In one embodiment, the other functional protein targets a subcellularregion. In one embodiment, the other functional protein has a PDZ or AISdomain.

Another aspect of the present invention is a vector for delivering aLACC protein comprising; a nucleic acid sequence that codes for LACCprotein and a promoter. In one embodiment the vector comprises a virus.A preferred embodiment of a virus is a lentivirus or retrovirus.

Another aspect of the present invention is a cell that expresses a LACCprotein. In one embodiment, the cell is a mammal cell, a stem cell, or aneuron.

Another aspect of the present invention is a cell line that expresses aLACC protein. In one embodiment, the cell line is a mammal cell line, astem cell line, or a neuronal cell line.

Another aspect of the present invention is a transgenic animal thatexpresses a LACC protein. In one embodiment, the transgenic animal is afly, worm, mouse, or zebrafish.

Another aspect of the present invention is a method of opticallycontrolling cell properties comprising; causing the cell to expresslight activated cation channel protein; and exposing the cell to lightto activate the LACC protein. In one preferred embodiment, the exposingof the cell to light creates an electrical response in the cell. Inanother preferred embodiment, the activation of the LACC protein causesthe release of a peptide (e.g. insulin, leptin, neuropeptide Y,substance P, human growth hormone, secretin, glucagon, endorphin,oxytocin, vasopressin, or orexin/hypocretin). In another preferredembodiment, the activation of the LACC protein causes the release of asmall molecule whose synthesis or release is dependent on cellelectrical activity (e.g., nitric oxide, or a cannabinoid such asanandamide or 2-arachidonylglycerol (2-AG)). In another preferredembodiment of the invention, the activation of the LACC protein causesthe release of a cytokine. In another preferred embodiment of theinvention, the cell causes a muscle cell, and the activation of the LACCprotein comprises causing the muscle cell to contract

Another aspect of the present invention is a method of controllingsynaptic transmissions comprising; causing a neuron that ends in asynapse to express a LACC protein; and exposing the neuron to light toactivate the LACC protein, wherein the exposing to light causes asynaptic event. In one embodiment, the synaptic event creates a synaptictransmission. In another embodiment, the synaptic transmission transmitsa signal to a second neuron. In another embodiment, the synaptictransmission drives activity through a connected neural network. In anembodiment, the synaptic event is excitatory or inhibitory. In yetanother embodiment, the synaptic event comprises the release of asmall-molecule neuromodulator such as norepinephrine, serotonin,dopamine, acetylcholine, D-serine, histamine, or other small moleculesthat modulate cellular function.

Another aspect of the present invention is a method for targeteddelivery of light-activated cation-channel proteins to specific cellscomprising; contacting said cells with a vector comprising a nucleicacid sequence comprising a LACC protein wherein said vector selectivelytargets specific cells.

Another aspect of the present invention is a method for targeteddelivery of light-activated cation-channel proteins to specific cellscomprising; contacting said cells with a vector comprising a nucleicacid sequence comprising a LACC protein and a cell specific promoter,wherein said specific cells express said LACC protein.

Another aspect of the present invention is a method for screening fordrugs comprising; expressing a LACC protein in a group of cells;exposing said group of cells to a compound that may have an effect onthe cells; exposing said groups of cells to light; and monitoring theelectrical response of cells within the group of cells to determinewhether or not the compound has an effect on the cells. In a preferredembodiment, the monitoring of electrical response comprises opticalimaging of fluorescence changes.

Another aspect of the present invention is a method for treating asubject comprising; delivering a vector comprising a LACC protein toexcitable cells within the subject; and exposing said cells to light toaffect such excitable cells.

Another aspect of the present invention is a method of controlling thebehavior of an organism comprising; delivering a vector comprising aLACC protein to cells within the organism; and exposing said cells tolight to control the organism's behavior.

Another aspect of the present invention is a method of stimulatingsubsets of nerve cells in the presence of other nerve cells comprising;exposing a group of nerve cells to a vector capable of geneticallytargeting a subset of the group of cells; and exposing the cells tolight to activate the LACC protein in the subset of cells.

Another aspect of the present invention is method of drivingdifferentiation in cells comprising: causing cells to express a LACCprotein; and exposing the cells to light to activate the LACC protein,wherein the activation of the LACC protein drives differentiation of theprogeny of the exposed cells. In a preferred embodiment, the cellscomprise stem cells.

In preferred embodiments described herein, the light-activated cationchannel protein is coded by a sequence of SEQ ID No. 2 or SEQ ID No. 3.

Another aspect of the invention is a method of treating a subjectcomprising administering to a patient in need thereof a therapeuticallyeffective amount of a light activated cation channel protein. Themethods described herein can be used for therapeutic and/or prophylaticbenefit. Preferably the light activated cation channel is administeredeither in the form of a cell, the cell expressing a light activatedcation channel protein or in the form of a vector comprising a nucleicacid sequence coding a light-activated cation channel protein, whereinthe administration of the vector causes the expression of the lightactivated cation channel protein in a cell in said patient. Preferably,the light-activated cation channel protein is coded by a sequence of SEQID No. 2 or SEQ ID No. 3. In preferred embodiments the activation of thelight-activated cation channel protein with light causes a release of apeptide, such as insulin, leptin, neuropeptide Y, substance P, humangrowth hormone, secretin, glucagon, endorphin, oxytocin, vasopressin,orexin/hypocretin, or a combination thereof. The activation of thelight-activated cation channel protein can also cause release of a smallmolecule, such as nitric oxide or a cannabinoid. Also, the activation ofthe light-activated cation channel protein can cause release of acytokine. In some embodiments, the light activated cation channel isexpressed in a muscle cell and activation of the light-activated cationchannel protein causes the muscle cell to contract. In otherembodiments, the light activated cation channel is expressed in aneuronal cell and activation of the light-activated cation channelprotein causes a synaptic event. The synaptic event can cause therelease of a small-molecule neuromodulator, such as norepinephrine,serotonin, dopamine, acetylcholine, D-serine, histamine, or acombination thereof. The synaptic event can be excitatory or inhibitory.The light activated cation channel can be expressed in a stem cell, suchas human embryonic stem cell, and activation of the light-activatedcation channel protein can cause the stem cell to differentiate. Thelight activated cation channel is expressed in a cancer cell andactivation of the light-activated cation channel protein causes amodulation of replication, survival, and/or controlled death in saidcancer cell. Preferably, the light-activated cation channel protein isChR2, Chop2, ChR2-310, or Chop2-310. An embodiment of an in vivo or exvivo method comprises expressing a light activated cation channelprotein in a stem cell, where said light activated cation channelprotein is activated by exposure to light and said activation causes amodulation of replication said stem cell. In preferred embodiments, thelight activated cation channel is expressed in retinal ganglion cells orspinal ganglion cells. A preferred embodiment is prosthetic devices,i.e., cells for implantation which express light activated cationchannel. Preferred prosthetics are for the eyes or ears and preferablyexpress ChR2, Chop2, ChR2-310, or Chop2-310. Preferably, the lightactivated cation channel protein is coded by a sequence of SEQ ID No. 2or SEQ ID No. 3.

Yet another preferred aspect is a method of predicting potential ionchannel modulating properties of a drug comprising: (a) expressing alight-activated cation channel protein and an ion channel of interest ina cell; (b) exposing said cell to light and monitoring a first responsein said cell; (c) exposing said cell to a candidate drug; (d) furtherexposing said cell to light and monitoring a second response in saidcell; and (e) determining an ion channel modulating property of saidcandidate drug based on a comparison of said first and second response.The monitoring of the responses can comprise optical imaging of changesin fluorescence in said cell or monitoring a signal transductionpathway. The signal transduction pathway can be monitored with anantibody, fluorescent small molecule, or a genetically encodedindicator. Preferably the light-activated cation channel protein isChR2, Chop2, ChR2-310, or Chop2-310. Preferably, the light-activatedcation channel protein is coded by a sequence of SEQ ID No. 2 or SEQ IDNo. 3.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1(a) shows a micrograph of hippocampal neurons expressing ChR2-YFP.

FIG. 1(b) shows b, Inward current in voltage-clamped neuron (left)evoked by 1 second of excitation light (indicated by lines throughoutfigures), with population data (middle; mean+standard deviation plottedthroughout figures; n=18). The figure also shows that light through aYFP filter evokes smaller currents (right; n=5).

FIG. 1(c) shows currents in a hippocampal neuron illuminated as in FIG.1(b), in response to light pulses.

FIG. 1(d) has voltage traces showing membrane depolarization and spikesin a current-clamped hippocampal neuron (left) evoked by 1 secondperiods of blue light. Right, properties of the first spike elicited(n=10), showing latency to spike threshold, latency to spike peak, andjitter of spike peak time.

FIG. 1(e) has voltage traces in response to brief light pulse sequences,with pulses lasting 5 ms (top), 10 ms (middle), and 15 ms (bottom).

FIG. 2(a) has voltage traces showing spikes in a current-clampedhippocampal neuron, in response to three deliveries of a Poisson trainof light pulses.

FIG. 2(b) shows trial-to-trial reliability of light evoked spike trains,as measured by comparing the presence or absence of a spike in tworepeated trials of the same Poisson train delivered to the same neuron.

FIG. 2(c) shows trial-to-trial jitter of the light-evoked spike trains.

FIG. 2(d) shows the percent fidelity of spike transmission throughoutthe entire 8-second Poisson train.

FIG. 2(e) shows latency of the spikes throughout each light pulsesequence (i), and jitter of spike times throughout the train (ii).

FIG. 2(f) has voltage traces showing spikes in three differenthippocampal neurons, in response to the same temporally patterned lightstimulus as used in FIG. 2(a).

FIG. 2(g) has a histogram showing how many of the 7 neurons spiked inresponse to each light pulse in the Poisson train.

FIG. 2(h) shows neuron-to-neuron jitter of spikes evoked by lightstimulation.

FIG. 3(a) has voltage traces showing spikes in a current-clampedhippocampal neuron evoked by 5, 10, 20, or 30 Hz trains of light pulses.

FIG. 3(b) has population data showing the number of spikes (out of 20possible) evoked in current-clamped hippocampal neurons.

FIG. 3(c) shows the number of extraneous spikes evoked by the trains oflight pulses, for the experiment described in FIG. 3(b).

FIG. 3(d) shows jitter of spike times throughout the train of lightpulses for the experiment described in FIG. 3(b).

FIG. 3(e) shows the latency to spike peak throughout the light pulsetrain for the experiment described in FIG. 3(b).

FIG. 4(a) has voltage traces showing subthreshold depolarizations in acurrent-clamped hippocampal neuron (left).

FIG. 4(b) illustrates how longer light pulses induced repeatabledepolarizations. Right, coefficients of variation (CV) for the inducedvoltage changes (n=5 neurons).

FIG. 4(c) shows excitatory synaptic transmission driven by light pulses.The glutamatergic blocker NBQX abolishes these synaptic responses(right).

FIG. 4(d) shows inhibitory synaptic transmission driven by light pulses.The GABAergic transmission blocker gabazine abolishes these synapticresponses (right).

FIG. 5(a) shows membrane resistance of neurons expressing ChR2 (CHR2+;n=18), not expressing ChR2 (CHR2−; n=18), or expressing ChR2 andmeasured 24 hours after exposure to a light-pulse protocol (CHR2+finished; n=12).

FIG. 5(b) shows membrane resting potential of the same neurons describedin FIG. 5(a).

FIG. 5(c) shows the number of spikes evoked by a 300-pA depolarisation,in the same neurons.

FIG. 6 shows a map of a retroviruses containing ChR2.

FIG. 7 shows a micrograph of a clonal stem cell line expressing ChR2introduced by lentiviral transduction.

FIG. 8 illustrates increased nuclear Ser-133 CREB phosphorylation inneural progenitor cell (NPC) triggered by light.

FIG. 9 is a micrograph of living zebrafish with ChR2 expressed in aneuron (left) and muscle cell (right).

FIG. 10 illustrates an embodiment of one of the methods of theinvention.

FIG. 11 illustrates an embodiment of one of the methods of theinvention.

FIG. 12 illustrates SEQ ID. No. 1.

FIG. 13 illustrates SEQ ID. No. 2.

FIG. 14 illustrates SEQ ID. No. 3.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel compositions and methods forcontrolling the electrical and chemical properties of cell membranesusing light. The present invention also provides for a noninvasive,genetically targeted, high temporal resolution control of membraneelectrical and chemical properties.

LACC

In a preferred embodiment of the invention, the light-activated cationchannel protein comprises a 7-transmembrane protein.

In a preferred embodiment of the invention, the LACC comprises theprotein, or portions of the protein Channelrhodopsin-2 (ChR2). ChR2 is arhodopsin derived from the unicellular green alga Chlamydomonasreinhardtii. The term “rhodopsin” as used herein is a protein thatcomprises at least two building blocks, an opsin protein, and acovalently bound cofactor, usually retinal (retinaldehyde). Therhodopsin ChR2 is derived from the opsin Channelopsin-2 (Chop2) (Nagel,et. al. Proc. Natl. Acad. Sci. USA 100:13940, and references citedtherein). The LACC protein of the present invention can incorporateretinal that is added to the system, or, depending on the cell type thatis used, background levels of retinal present in the cell may producethe required retinal. It is intended herein that the methods of theinvention encompass either the opsin or the rhodopsin form of the LACCprotein, e.g. Chop2 or ChR2. Typically, Chop2 and ChR2 can beinterconverted by the addition or removal of the cofactor. Thus, as usedherein, a LACC protein comprises an opsin with or without a co-factor.For example, as used herein, where a nucleic acid codes for an opsinprotein such as Chop2, it codes for a light activated cation channelprotein such as ChR2. Additionally, as used herein, where a cellexpresses an opsin protein such as Chop2, it expresses a LACC protein.

The LACC of the present invention may also cause the modulation of theflow of anions such as chloride across a membrane when activated bylight. Optically induced electrical and chemical changes due toactivation of the LACC by light are also included within the invention.

In some embodiments of the invention it is desirable to add cofactor(usually in the nanomolar to micromolar range). In other embodiments, noaddition of retinal is required. In some cases, the medium may providethe required cofactor. In a preferred embodiment of the presentinvention, the LACC protein covalently binds retinal. The term retinal,as used in comprises all-trans retinal, 11-cis retinal, and otherisomers of retinal.

In some embodiments of the invention the protein Bcdo can be expressedalong with ChR2. Bcdo converts the common dietary molecule beta caroteneinto retinal (Yan et. al., Genomics 72 (2):193 (2001)), thus providingretinal to convert Chop2 to ChR2.

As used herein, the terms “ChR2” and “Chop2” mean the full proteins orfragments thereof. A preferred embodiment of the present inventioncomprises the amino terminal 310 amino acids of Chop2 which is referredto herein as Chop2-310. A preferred embodiment the present inventioncomprises the amino terminal 310 amino acids of ChR2 which is referredto as ChR2-310. The amino-terminal 310 amino acids of ChR2 show homologyto the 7-transmembrane structure of many microbial-type rhodopsins, andcomprise a channel with a light-gated conductance. In an embodiment ofthe invention, a LACC protein comprises a 7-transmembrane protein.Preferably the LACC protein is a 7-transmembrane protein that either hasa binding affinity for retinal, or has retinal bound to it.

In a preferred embodiment, the LACC of the present invention is derivedfrom a microbial-type rhodopsin. In a preferred embodiment, the LACC ofthe present invention is derived from a bacteriorhodopsin.

One aspect of the present invention is a single-component protein thatis a LACC protein. As used herein, a single component protein is asingle covalently linked chain of amino acids. Multiple componentsystems require communication between non-covalently linked molecules,which can be much slower than within-protein signaling viaconformational changes. Unlike previous methods requiring severalprotein components, the present invention allows the creation of lightgated membrane conductance with a single protein component. While notbeing bound by theory, it is believed that the retinal in ChR2, as amicrobial type rhodopsin, is strongly bound, allowing the retinal tore-isomerize to the all-trans ground state in a dark reaction withoutthe need for other enzymes. This mechanism allows for fast recovery(closing of the ionic channel) when the light is removed, and itobviates the need for other enzyme components for re-generation of theall trans-retinal and closing of the channel.

In a preferred embodiment of the invention the light-activatedcation-channel Channelrhodopsin-2 (ChR2) is genetically introduced intoa cellular membrane.

The LACC protein of the present invention also comprises the proteinsequence of Chop2-310 [SEQ ID NO:1, depicted in FIG. 12]. “Protein” inthis sense includes proteins, polypeptides, and peptides. Also includedwithin the LACC protein of the present invention are amino acid variantsof the naturally occurring sequences, as determined herein. Preferably,the variants are greater than about 75% homologous to the proteinsequence of Chop2 or Chop2-310, more preferably greater than about 80%,even more preferably greater than about 85% and most preferably greaterthan 90%. In some embodiments the homology will be as high as about 93to about 95 or about 98%. Homology in this context means sequencesimilarity or identity, with identity being preferred. This homologywill be determined using standard techniques known in the art. Thecompositions of the present invention include the protein and nucleicacid sequences provided herein including variants which are more thanabout 50% homologous to the provided sequence, more than about 55%homologous to the provided sequence, more than about 60% homologous tothe provided sequence, more than about 65% homologous to the providedsequence, more than about 70% homologous to the provided sequence, morethan about 75% homologous to the provided sequence, more than about 80%homologous to the provided sequence, more than about 85% homologous tothe provided sequence, more than about 90% homologous to the providedsequence, or more than about 95% homologous to the provided sequence.

LACC proteins of the present invention may be shorter or longer than theprotein sequence of Chop2 or Chop2-310. Thus, in a preferred embodiment,included within the definition of LACC proteins are portions orfragments of the protein sequence of Chop2 or of Chop2-310. In addition,nucleic acids of the invention may be used to obtain additional codingregions, and thus additional protein sequence, using techniques known inthe art.

In a preferred embodiment, the LACC proteins of the present inventionare derivative or variant protein sequences, as compared to Chop2 orChop2-310. That is, the derivative LACC proteins of the presentinvention will contain at least one amino acid substitution, deletion orinsertion, with amino acid substitutions being particularly preferred.The amino acid substitution, insertion or deletion may occur at anyresidue within the LACC protein.

Also included in an embodiment of the LACC proteins of the presentinvention are amino acid sequence variants of the ChR2, Chop2, ChR2-310,Chop-310 or [SEQ ID NO:1, depicted in FIG. 12]. These variants fall intoone or more of three classes: substitutional, insertional or deletionalvariants. These variants ordinarily are prepared by site specificmutagenesis of nucleotides in the DNA encoding the LACC proteins, usingcassette or PCR mutagenesis or other techniques well known in the art,to produce DNA encoding the variant, and thereafter expressing the DNAin recombinant cell culture. Amino acid sequence variants arecharacterized by the predetermined nature of the variation, a featurethat sets them apart from naturally occurring allelic or interspeciesvariation of the LACC proteins of the present invention. The variantstypically exhibit the same qualitative biological activity as thenaturally occurring analogue, although variants can also be selectedwhich have modified characteristics.

While the site or region for introducing an amino acid sequencevariation is predetermined, the mutation per se need not bepredetermined. For example, in order to optimize the performance of amutation at a given site, random mutagenesis may be conducted at thetarget codon or region and the expressed breast cancer variants screenedfor the optimal combination of desired activity. Techniques for makingsubstitution mutations at predetermined sites in DNA having a knownsequence are well known, for example, M13 primer mutagenesis and PCRmutagenesis.

Amino acid substitutions are typically of single residues; insertionsusually will be on the order of from about 1 to 20 amino acids, althoughconsiderably larger insertions may be tolerated. Deletions range fromabout 1 to about 20 residues, although in some cases deletions may bemuch larger.

Substitutions, deletions, insertions or any combination thereof may beused to arrive at a final derivative. Generally these changes are doneon a few amino acids to minimize the alteration of the molecule.However, larger changes may be tolerated in certain circumstances. Insome embodiments, small alterations in the characteristics of the LACCproteins of the present invention are desired, substitutions aregenerally made in accordance with the following table:

TABLE I Original Exemplary Residue Substitutions Ala Ser Arg Lys AsnGln, His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn, Gln Ile, Leu,Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe Met, Leu, Tyr SerThr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu

Substantial changes in function are made by selecting substitutions thatare less conservative than those shown in Table 1. For example,substitutions may be made which more significantly affect the structureof the polypeptide backbone in the area of the alteration, for examplethe alpha-helical or beta-sheet structure; the charge or hydrophobicityof the molecule at the target site; or the bulk of the side chain. Thesubstitutions which in general are expected to produce the greatestchanges in the polypeptide's properties are those in which (a) ahydrophilic residue, e.g. seryl or threonyl is substituted for (or by) ahydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl oralanyl; (b) a cysteine or proline is substituted for (or by) any otherresidue; (c) a residue having an electropositive side chain, e.g. lysyl,arginyl, or histidyl, is substituted for (or by) an electronegativeresidue, e.g. glutamyl or aspartyl; or (d) a residue having a bulky sidechain, e.g. phenylalanine, is substituted for (or by) one not having aside chain, e.g. glycine.

The variants or derivatives typically exhibit the same qualitativeactivity as the Chop2, ChR2, Chop-310, or ChR2-310 protein, althoughvariants or derivatives also are selected to modify the characteristicsof the LACC proteins as needed. Variants or derivatives can showenhanced ion selectivity, stability, speed, compatibility, and reducedtoxicity. For example, the protein can be modified such that it can bedriven by different wavelength of light than the wavelength of around460 nm of the wild type ChR2 protein. The protein can be modified, forexample, such that it can be driven at a higher wavelength such as about480 nm, 490 nm, 500 nm, 510 nm, 520 nm, 530 nm, 540 nm, 550 nm, 560 nm,570 nm, 580 nm, or 590 nm.

The LACC proteins of the present invention can incorporate un-naturalamino acids as well as natural amino acids. The unnatural amino acidscan be used to enhance ion selectivity, stability, speed, compatibility,or to lower toxicity.

An aspect of the present invention is a fusion protein comprising alight-activated cation channel protein. It is well known in the art thatfusion proteins can be made that will create a single protein with thecombined activities of several proteins. In one embodiment, the fusionproteins can be used to target Chop2 or ChR2 to specific cells orregions within cells.

One embodiment of a fusion protein comprising a LACC protein is a fusionprotein that targets sub-cellular regions of the cell. The fusionproteins can target, for instance, axons, dendrites, and synapses ofneurons. In one preferred embodiment, a PDZ (PSD-95, Dlg and ZO-1)domain is fused to ChR2 or Chop2 which target dendrites. In anotherpreferred embodiment, Axon initial segment (AIS) domain is fused to ChR2or Chop2 which target axons.

Other fusion proteins of the present invention are proteins combiningChR2 and a fluorescent protein in order to allow for monitoring of thelocalization of ChR2. Preferred fusion proteins are those with redfluorescent protein (mCherry), yellow fluorescent protein (YFP), cyanfluorescent protein (CFP) and green fluorescent protein (GFP). Thesefusion proteins, such as the ChR2-mCherrry fusion protein, allow for theindependent stimulation of ChR2 and the simultaneous monitoring oflocalization. The simultaneous stimulation and monitoring oflocalization can be carried out in many cell types including mammaliansystems.

It is an aspect of the invention to provide a light-activated cationchannel protein that is non-toxic in the cells in which it is expressed.Preferably, the light-activated ion channel proteins of the presentinvention do not perturb the basal electrical properties, alter thedynamic electrical properties, or jeopardize the prospects for cellularsurvival. Preferably, the light-activated cation channel proteins of thepresent invention do not alter the membrane resistance of the cells inthe absence of light. Preferably, the light-activated ion channels donot lead to apoptosis in the cells, nor lead to the generation ofpyknotic nuclei. Preferably, in the absence of light, the presence ofthe LACC protein does not alter cell health or ongoing electricalactivity, at the level of subthreshold changes in voltage or in spikeoutput, either by shunting current through leaky channels or by alteringthe voltage dependence of existing neuronal input-output relationships.Preferably, the presence of LACC protein creates no significantlong-term plastic or homeostatic alterations in the electricalproperties of neurons expressing the protein.

Another aspect of the present invention provides nucleic acid sequenceswhich code for the LACC proteins of the present invention. It would beunderstood by a person of skill in the art that the LACC proteins of thepresent invention can be coded for by various nucleic acids. Each aminoacid in the protein is represented by one or more sets of 3 nucleicacids (codons). Since many amino acids are represented by more than onecodon, there is not a unique nucleic acid sequence that codes for agiven protein. It is well understood by persons of skill in the art howto make a nucleic acid that can code for the LACC proteins of thepresent invention by knowing the amino acid sequence of the protein. Anucleic acid sequence that codes for a polypeptide or protein is the“gene” of that polypeptide or protein. A gene can be RNA, DNA, or othernucleic acid than will code for the polypeptide or protein.

One preferred embodiment of a nucleic acid sequence comprises [SEQ IDNO:2, depicted in FIG. 13].

It is known by persons of skill in the art that the codon systems indifferent organisms can be slightly different, and that therefore wherethe expression of a given protein from a given organism is desired, thenucleic acid sequence can be modified for expression within thatorganism.

An aspect of the present invention provides a nucleic acid sequence thatcodes for a light-activated cation protein that is optimized forexpression with a mammalian cell. A preferred embodiment comprises anucleic acid sequence optimized for expression in a human cell.

A preferred embodiment of a nucleic acid sequence that codes for alight-activated cation protein that is optimized for expression with ahuman cell comprises [SEQ ID NO:3, depicted in FIG. 14].

Another aspect of the present invention provides for reagents forgenetically targeted expression of the LACC proteins including ChR2.Genetic targeting can be used to deliver light-activated cation channelproteins to specific cell types, to specific cell subtypes, to specificspatial regions within an organism, and to sub-cellular regions within acell. Genetic targeting also relates to the control of the amount oflight-activated cation channel protein expressed, and the timing of theexpression.

A preferred embodiment of a reagent for genetically targeted expressionof the LACC protein comprises a vector which contains the gene for theLACC protein.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting between different genetic environments anothernucleic acid to which it has been operatively linked. The term “vector”also refers to a virus or organism that is capable of transporting thenucleic acid molecule. One type of preferred vector is an episome, i.e.,a nucleic acid molecule capable of extra-chromosomal replication.Preferred vectors are those capable of autonomous replication and/orexpression of nucleic acids to which they are linked Vectors capable ofdirecting the expression of genes to which they are operatively linkedare referred to herein as “expression vectors”. Other preferred vectorsare viruses such as lentiviruses, retroviruses, adenoviruses and phages.Preferred vectors can genetically insert LACC proteins into bothdividing and non-dividing cells. Preferred vectors can geneticallyinsert LACC proteins in-vivo or in-vitro.

Those vectors that include a prokaryotic replicon can also include aprokaryotic promoter capable of directing the expression (transcriptionand translation) of the LACC protein in a bacterial host cell, such asE. coli. A promoter is an expression control element formed by a DNAsequence that permits binding of RNA polymerase and transcription tooccur. Promoter sequences compatible with bacterial hosts are typicallyprovided in plasmid vectors containing convenience restriction sites forinsertion of a DNA segment of the present invention. Typical of suchvector plasmids are pUC8, pUC9, pBR322, and pBR329 available from BioRadLaboratories, (Richmond, Calif.) and pPL and pKK223 available fromPharmacia, (Piscataway, N.J.).

Expression vectors compatible with eukaryotic cells, can also be used.Eukaryotic cell expression vectors are well known in the art and areavailable from several commercial sources. Typically, such vectors areprovided containing convenient restriction sites for insertion of thedesired DNA homologue. Typical of such vectors are pKSV-10 (Pharmacia),pBPV-1/PML2d (International Biotechnologies, Inc.), and pTDT1 (ATCC, No.31255).

One preferred embodiment of an expression vector of the presentinvention is a lentivirus comprising the gene for ChR2 or Chop2 and anEF1-alpha promoter. This lentivirus vector is used in one aspect of thepresent invention to create stable cell lines. The term “cell line” asused herein is an established cell culture that will continue toproliferate given the appropriate medium.

One preferred embodiment of an expression vector of the presentinvention is a lentivirus comprising the gene for ChR2 or Chop2 and acell specific promoter. Examples of cell specific promoters arepromoters for somatostatin, parvalbumin, GABAα6, L7, and calbindin.Other cell specific promoters are promoters for kinases such as PKC,PKA, and CaMKII; promoters for other ligand receptors such as NMDAR1,NMDAR2B, GluR2; promoters for ion channels including calcium channels,potassium channels, chloride channels, and sodium channels; andpromoters for other markers that label classical mature and dividingcell types, such as calretinin, nestin, and beta3-tubulin.

Another preferred embodiment is a lentivirus containing tetracyclineelements that allow control of the gene expression levels of ChR2,simply by altering levels of exogenous drugs such as doxycycline. Thismethod, or other methods that place ChR2 under the control of adrug-dependent promoter, will enable control of the dosage of ChR2 incells, allowing a given amount of light to have different effects onelectrical activation, substance release, or cellular development

One aspect of the invention is nucleic acid sequences comprising thegene for LACC proteins and promoters for genetically targeted expressionof the proteins. The genetically targeted expression of the LACCs of thepresent invention can be facilitated by the selection of promoters. Theterm “promoter” as used herein is nucleic acid sequence that enables aspecific gene to be transcribed. The promoter usually resides near aregion of DNA to be transcribed. The promoter is usually recognized byan RNA polymerase, which, under the control of the promoter, createsRNA, which is then converted into the protein for which it codes. By useof the appropriate promoter, the level of expression of LACC protein canbe controlled. Cells use promoters to control where, when, and how muchof a specific protein is expressed. Therefore, by selecting a promoterthat is selectively expressed predominantly within one type of cell, onesubtype of cells, a given spatial region within an organism, orsub-cellular region within a cell, the control of expression of LACC canbe controlled accordingly. The use of promoters also allows the controlof the amount of LACC expressed, and the timing of the expression. Thepromoters can be prokaryotic or eukaryotic promoters.

One embodiment of the invention is a nucleic acid sequence comprisingthe gene for LACC protein and a general purpose promoter. A generalpurpose promoter allows expression of the LACC protein in a wide varietyof cell types. One example of a general purpose promoter of the presentinvention is The EF1-alpha promoter. The EF-1 alpha gene encodes forelongation factor-1 alpha which is one of the most abundant proteins ineukaryotic cells and is expressed in almost all kinds of mammaliancells. The promoter of this “housekeeping” gene can lead to persistentexpression of the transgene in vivo. Another preferred general promoteris the CMV (cytomegalovirus) promoter, which can drive gene expressionat very high levels. Still other preferred general-purpose promotersinclude those for CaMKII and synapsin I (Dittgen et. al,PNAS101:18206-11 (2004)).

One embodiment of the present invention is a nucleic acid sequencecomprising the gene for LACC protein and a cell specific promoter.Examples of cell specific promoters are promoters for somatostatin,parvalbumin, GABAα6, L7, and calbindin. Other cell specific promotersare promoters for kinases such as PKC, PKA, and CaMKII; promoters forother ligand receptors such as NMDAR1, NMDAR2B, GluR2; promoters for ionchannels including calcium channels, potassium channels, chloridechannels, and sodium channels; and promoters for other markers thatlabel classical mature and dividing cell types, such as calretinin,nestin, and beta3-tubulin. In a preferred embodiment of the presentinvention, the nucleic acid comprises a bacterial artificial chromosome(BAC).

One embodiment of the present invention is promoter is an induciblepromoter. For instance, the promoter can be inducible by a trans-actingfactor which responds to an exogenously administered drug. The promoterscould be, but are not limited to tetracycline-on or tetracycline-off, ortamoxifen-inducible Cre-ER.

Cells

One aspect of the present invention is a cell that expresses LACCproteins, and specifically a cell that expresses ChR2 or Chop2. Anotheraspect of the invention is a LACC protein expressing-cell that alsoexpresses other ion channels, receptors, or signaling proteins, both inthe normal and/or impaired form. Another aspect to the invention is abusiness method to make commercially available the cells of theinvention.

The cells of the present invention can be created using a vectorincluding a DNA expression vector, a virus or an organism. Preferredvectors include lentiviruses and retroviruses. In some cases, inparticular where robust cell lines are involved, expression of ChR2 canbe induced by using lipofection techniques, such as exposing cell linesto micelles containing Lipofectamine or Fugene, and then FACS-sorting toisolate stably expressing cell lines.

Cells of any origin, preferably those cells that are capable of growthin tissue culture, are candidate cells for transfection or infectionwith a LACC protein such as ChR2 or Chop2. Non-limiting examples ofspecific cell types that can be grown in culture include connectivetissue elements such as fibroblast, skeletal tissue (bone andcartilage), skeletal, cardiac and smooth muscle, epithelial tissues(e.g. liver, lung, breast, skin, bladder and kidney), neural cells (gliaand neurones), endocrine cells (adrenal, pituitary, pancreatic isletcells), bone marrow cells, melanocytes, and many different types ofhematopoetic cells. Suitable cells can also be cells representative of aspecific body tissue from a subject. The types of body tissues include,but are not limited, to blood, muscle, nerve, brain, heart, lung, liver,pancreas, spleen, thymus, esophagus, stomach, intestine, kidney, testis,ovary, hair, skin, bone, breast, uterus, bladder, spinal cord andvarious kinds of body fluids. Cells in culture can be freshly isolatedfrom body tissues (known as primary culture) or subcultured by expansionand/or cloning of the cells present in the primary culture (known ascell lines).

Cells of different developmental stages (embryonic or adult) of anorganism, or more specifically of various developmental originsincluding ectoderm, endoderm and mesoderm, can also be applied. Anothertype of cells embodied in the present invention is a “personal celltype”, which comprises cells derived from individuals of a family, orindividuals from different generations within the same pedigree.

Of particular interest are cells that are associated with a particulardisease or with a specific disease stage, cells derived from natural andinduced immune deficiency states, cardiovascular disease, neuronaldisease, inflammation states and diseases caused by a variety ofpathogens. The association with a particular disease or disease stagemay be established by the cell's aberrant behavior in one or morebiological processes such as cell cycle regulation, celldifferentiation, apoptosis, chemotaxis, cell motility and cytoskeletalrearrangement. A disease cell may also be confirmed by the presence of apathogen causing the disease of concern (e.g. HIV for AIDS and HBV forhepatitis B).

Preferred cells are mammalian cells and cell lines derived frommammalian cells. Other preferred cells are embryonic stem cells andadult stem cells including hematopoetic stem cells, bone marrow, neuralstem cells, epithelial stem cells, skin stem cells. Preferred cell linesappropriate for ChR2 expression include, HEK cells, neural stem celllines, pancreatic islet cell lines, and other excitable or secretorycells.

The LACC proteins, when activated by light can also cause other ionchannel proteins within the membrane to be activated. Thus, an aspect ofthe invention is a cell that has both the LACC protein and other ionchannels or a splice variant of an ion channel that can be activated bythe LACC protein. Examples of ion channels include, but are not limitedto, Voltage-gated channels such as sodium and potassium voltage-gatedchannels of nerve and muscle; Ligand-gated such as Acetylcholinereceptor, AMPA receptor and other neurotransmitter-gated channels;Cyclic nucleotide-gated channels such as calcium-activated channels;cardiac ion channels such as HERG channels; Stretch-activated channels;G-protein-gated channels; Inward-rectifier K channels; Resting channels;Store-operated channels such as calcium release-activated calcium (CRAC)channel; as well as other calcium channels, other potassium and sodiumchannels. The one or more ion channel proteins can be artificial, andcan be targeted to be expressed in the cell containing the LACC protein.

A preferred embodiment of the invention is a cell that comprises a LACCprotein and one or more ion channel proteins that can be activated andthus controlled and whose behavior can be understood. Another preferredembodiment of the invention is a cell that comprises a LACC protein andone or more ion channel proteins that can be activated and thuscontrolled for screening for ion channel modulators as described below.Another preferred embodiment of the invention is a cell that comprises aLACC protein and one or more cardiac ion channels (e.g. HERG channel)that can be activated and thus controlled for screening for side effectof compounds as described below. Another preferred embodiment is anarticle of manufacture as described below containing a cell thatcomprises a LACC protein and one or more ion channel proteins that canbe activated as described by the method herein.

A preferred cell that expresses LACC proteins, and specifically a cellthat expresses ChR2 or Chop2 is a mammalian cell.

One aspect of the present invention is a cell line that expresses LACCproteins, and specifically a cell line that expresses ChR2 or Chop2. Thecell line of the present invention can be created using a vectorincluding a DNA expression vector, a virus or an organism. Preferredvectors include lentiviruses and retroviruses. In some cases, inparticular where robust cell lines are involved, expression of ChR2 canbe induced by using lipofection techniques, such as exposing cell linesto micelles containing Lipofectamine or Fugene, and then FACS-sorting toisolate stably expressing cell lines. Preferred cell lines are mammaliancell lines as described above. Preferred cell lines are neuronal celllines or other excitable cell lines. Preferred cell lines appropriatefor ChR2 expression include HEK cells, neural stem cell lines,pancreatic islet cell lines, and other excitable or secretory cells.

A preferred cell line of the present invention is a clonal neuronal stemcell line expressing ChR2 under the EF1-alpha promoter. Such a cell lineis useful for the screening of drugs, particularly drugs that affect theinfluence of electrical activity on neuronal genesis, development, andapoptosis.

Another preferred cell line of the present invention is a line ofhippocampal neurons that expresses ChR2 under the EF1-alpha promoter.Such a cell line is useful for the in-vitro screening of drugs, and as amodel for controlling neurons with light in-vivo.

An embodiment of the invention is a stem cell lines that providetherapies based on transplantable, optically activatable cells thatrelease substances as described below such as insulin, growth hormones,or other small molecules or polypeptides. Control of substance releasewith the present invention can be done on the second-to-minutetimescale, allowing precise management of drug dosing, especially forconditions like diabetes or growth retardation.

The cells of the invention can be grown as a monolayer anchored onto asolid phase substrate, or as aggregates in a suspension culture. Thechoice of substrate is determined largely by the type of cell and thedesired growth parameters (e.g. growth rate, desired density, mediarequirements etc.) Most cells can be propagated on a substrate made ofe.g., glass, plastic or ceramic material. For certain cell types, suchas neurons, epithelial and muscle cells, substrates pre-coated withcharged substances that enhance cell attachment and spreading arepreferred. Commonly employed coating materials include biologicalsubstrates that bear a net positive charge. Non-limiting examples ofbiological substrates include extracellular matrix/adhesion proteinssuch as laminin, fibronectin, collagen, or synthetic polypeptide such aspoly-lysine. A variety of non-biological substrates such as membranesmade of nitrocellulose, nylon, polytetrafluoroethylene, or any otherimplant materials can also be used to support growth of cells in asuitable medium according to cell type.

Precautions are generally taken to maintain membrane integrity andpreserve cell membrane components when harvesting, cells cultured ondifferent substrates. The method of the invention contemplate the use oftraditional method of dissociating of anchored cells or cell layersincluding proteolytic enzymes such as serine proteinase, trypsin. Inaddition cells of the present invention can be removed from the culturesubstrates by agents that minimize damages to the cell surface antigens.These agents include chelating agents, such as EDTA and EGTA, which bindto divalent metal ions (e.g. calcium and magnesium) known to benecessary for cell-substrate attachment. Other suitable celldissociation agents encompass collagenases, dispases, and neutralproteinases when used in conjunction with serine proteinase inhibitors(e.g. soybean trypsin inhibitor). Treatment of cells with these agentsmostly results in disruption of the extracellular matrix componentswhile preserving the cell surface proteins. The time required to detachthe cells anchored on a solid substrate can vary depending on theprotease enzymes chosen, but will normally be a period of about 1 minuteto 30 minutes, and preferably about 5 minutes to 15 minutes. Theenzymatic treatment can be carried out at room temperature or at about37° C. Excess enzyme can be removed by gentle washing with buffershaving pH and salt concentrations in the physiological range that areroutinely prepared by one skilled in the art.

Cell viability may be confirmed by the measurement of membraneintegrity. The methods for assessing membrane integrity are known in theart. The most common assay involves staining cells with a dye thatreacts with either living or dead cells. As is apparent to one skilledin the art, exemplary dyes include trypan blue, eosin Y, naphthaleneblack, nigrosin, erythrosin B and fast green.

Transgenic Animals

One aspect of the invention is a transgenic animal that expresses a LACCprotein. A preferred embodiment is a transgenic animal that expressesChop2 or ChR2. Expression of LACC protein in particular subsets ofneurons can be used for analyzing circuit function, behavior,plasticity, and animal models of psychiatric disease

Preferred transgenic animal species of the present invention thatexpresses LACC protein include zebrafish (Danio rerio). In zebrafish,the LACC protein can be introduced fish embryos by acute injection atthe few-hundred cell stage.

Another preferred transgenic animal species of the present inventionthat expresses LACC protein is flies (Drosophila melanogaster). In onepreferred embodiment, flies express ChR2 under the UAS promoter for usein the GAL4-UAS system). Another preferred transgenic animal species ofthe present invention that expresses LACC protein is worms(Caenorhabditis elegans). In one preferred embodiment, stable lines aremade with injection of plasmids containing ChR2 under specific promotersinto the gonad.

Another preferred transgenic animal species of the present inventionthat expresses LACC protein is mice. In one preferred embodiment, micethat express LACC protein are made using BAC (bacterial artificialchromosome) transgenic technology, as well as position effectvariegation techniques.

One preferred embodiment of a transgenic animal of the present inventionthat expresses LACC protein is Drosophila in which ChR2 is expressed inserotonergic and dopaminergic neurons, which are important for thedriving of motivated behavior and the creation of finely tuned motorpatterns.

One preferred embodiment of a transgenic animal of the present inventionthat expresses LACC protein is Caenorhabditis elegans in which ChR2 isexpressed in serotonergic and dopaminergic neurons, which are importantfor the driving of motivated behavior and the creation of finely tunedmotor patterns.

Another preferred embodiment of the present invention is a transgenicanimal wherein the LACC is expressed under a specific promoter. Anotherpreferred embodiment of the present invention is a transgenic animalwherein the LACC expressed in the transgenic animal is introduced via aBAC. Another preferred embodiment of the present invention is atransgenic animal wherein the LACC gene is knocked into a known locus.

Channel Properties

In the present invention, electrical spikes, or action potentials, arecreated across a membrane by illumination with light. The light can beprovided by a light source such as a xenon lamp, or the light source canbe a laser. While a laser can be used, it would be understood by oneskilled in the art that if the intensity of light is too high, there canbe damaged to the cells under illumination due to local heating, etc. Itis preferred to use a light intensity that does not damage the cells. Ina preferred embodiment of the present invention medium intensity lightis used to activate the ion channel. A preferred level of light isbetween about 0.1 mW/mm² and about 500 mW/mm², preferably from about 1mW/mm² and about 100 mW/mm², and most preferably from about 5 mW/mm² andabout 50 mW/mm². In a preferred embodiment, the LACC protein is ChR2 andthe wavelength of the illuminating light is between about 400 nm andabout 600 nm, preferably from about 450 nm and about 550 nm, mostpreferably from about 450 nm and about 490 nm.

In accordance with the present invention, it has been discovered thatthe light-activated ion channels of the invention can generatemillisecond-timescale spikes when incorporated into neurons andilluminated with rapid pulses of light.

One aspect of the present invention is a light-activated ion channelthat is expressed in an excitable cell and will operate on themillisecond timeframe. In a preferred embodiment, a LACC responds within100 millisecond (ms), more preferably within 10 ms, even more preferablywithin 1 ms, and most preferably within 0.1 ms. A preferred embodimentof the present invention is ChR2 that is expressed in a neuron or otherexcitable cell.

In accordance with this invention, it has also been discovered that acell expressing a LACC of the present invention will reliably generatespikes on the millisecond timeframe.

In a preferred embodiment of a light-activated ion channel that isexpressed in an excitable cell and will operate on the millisecondtimeframe, the excitable cell responds in less than 10 ms, preferablyless than 5 ms, and most preferably within 1 ms of being illuminated.

One aspect of the present invention is a LACC protein expressed in acell membrane that provides stable photo-currents over long time scales.In a preferred embodiment the LACC protein is ChR2, and the photocurrentdoes not change by more than 20% over an hour of illumination,preferably not more than 10%, and most preferably not a detectablechange over an hour of illumination.

One aspect of the present invention is a LACC protein expressed in acell membrane that provides precisely timed spikes with low temporaljitter. The temporal jitter is preferably lower than 10 ms, morepreferably less than 5 ms, and most preferably lower than 3 ms whenmeasured within neurons.

One aspect of the present invention is a LACC protein expressed in acell membrane that provides low temporal jitter across neutrons. Lowtemporal jitter across neutrons is desirable because it allowsheterogeneous populations of neutrons to be controlled in concert.Preferably the temporal jitter across neutrons is also less than 10 ms,more preferably less than 5 ms, and most preferably lower than 3 ms.Preferably the across neutron jitter is within 50% of the within neutronjitter, most preferably the across neutron jitter is within 20% of thewithin neutron jitter, most preferably the within neutron jitter isindistinguishable from the across neutron jitter.

One aspect of the present invention is a LACC protein expressed in amembrane that provides reliable and precisely timed subthreshold pulses.

It has been discovered that a cell expressing a light-activated cationchannel protein of the present invention showed small trial-to-trialvariability in the subthreshold deflections evoked by repeated lightpulses. The light-activated cation channel proteins of the presentinvention such as ChR2 can therefore be employed to drive reliably timedsubthreshold depolarizations with precisely determined amplitude.

An aspect of the present invention is a LACC protein expressed in amembrane that has repeatable subthreshold polarizations. In a preferredembodiment of the invention, the LACC protein expressed in a membranegenerates subthreshold pulses with a coefficient of variation of lessthan 0.2 when measured over 5 consecutive pulses, more preferably thecoefficient of variation is less than 0.15 when measured over 5consecutive pulses, and most preferably the coefficient of variation isless than 0.10 when measured over 5 pulses.

The present invention allows for the optical control of cell propertiesby expressing a LACC protein within the cell, and activating the proteinwith light. The invention allows the optical control of excitable cells.One type of control is the control of electrical properties of the cellmembrane for processes such as control of neurons and of transmissionsof signals between neurons. Another type of control is control of influxof ions including Ca+2. The control of influx of Ca+2 is known to affectthousands of cellular processes. The optical control of the propertiesof excitable cells can be carried out in-vivo or in-vitro, and can beused for understanding biological processes, for drug discovery, and fortherapeutic uses.

Another type of control provided by the present invention is the use ofthe activation of the LACC protein to cause the cell to releaseproteins, peptides, or small molecules. In one embodiment of theinvention, through the light activation of LACC protein, the cell can becaused to release a protein, such as a cytokine. In one embodiment ofthe invention, through the light activation of ChR2, the cell can becaused to release a peptide such as insulin, leptin, neuropeptide Y,substance P, human growth hormone, secretin, glucagon, endorphin,oxytocin, vasopressin, or orexin/hypocretin. In another embodiment, theactivation of the LACC protein can cause the release of a small moleculewhose synthesis or release is dependent on electrical activity, such asnitric oxide, or a cannabinoid such as anandamide or2-arachidonylglycerol (2-AG).

The present invention also allows for the control of cellular activitybeyond the release of substances. For instance, LACC expressed in amuscle cell can allow for optical control of muscle cell contraction.This optical contraction of muscle cells, especially the contraction ofspecific muscle cells in the presence of other muscle cells, can be usedfor therapeutic purposes, or for the optical control of muscularcontractions.

The control provided by the present invention also included initiatingspecific intracellular signal conduction pathways. These pathwaysinclude, but are not limited to, kinases, transcription factors, andsecond messenger systems. These pathways can be specifically activatedby the present invention due to the specific temporal pattern of lightthat is used. The pathways can also be specifically activated due to thespecific sub-cellular localization of the LACC.

An aspect of the present invention is a method of optically controllinga cell comprising; causing the cell to express LACC protein; andilluminating the cell with light to activate the LACC protein.

A preferred embodiment of the method of optically controlling cellproperties is a method in which the LACC protein is ChR2.

Another preferred embodiment of the method of optically controlling cellproperties is a method in which the cells are neurons or other excitablecells. The neurons can be connected to other neural cells and can be ina neural network.

Another preferred embodiment of the method of optically controlling cellproperties is a method in which the cells are spatially or geneticallytargeted subsets of cells that express the LACC protein of the presentinvention and can be specifically activated.

Another preferred embodiment of a method of controlling cell propertiescomprises a method wherein the activation of the LACC also controlsother ion channels within the cell. For instance, a cell can be causedto express LACC and one or more other ion channel proteins which may beartificial proteins. The LACC activation can then be used to activate orinactivate the other ion channel proteins within the cell. In oneembodiment, a cell expressing LACC can be illuminated with light inorder to depolarize the cell membrane sufficiently to inactivate thecell's other ion channels.

One aspect of the invention is a method of driving cell differentiationusing light. LACC protein can be used to selectively targetdifferentiating cells whose cell fate is dependent on activity. Forexample, ChR2 can be delivered to a progenitor cell or cell line, andthen light can be used to drive the differentiation of the cell intoappropriate progeny.

In accordance with this invention, it has also been discovered that acell expressing a light-activated cation channel protein of the presentinvention will reliably generate spikes on the millisecond timeframe ifthe optical illuminations is provided in pulses in which a rapid lightpulse is followed by a period of darkness.

In a preferred embodiment of the method of optically controlling cellproperties is a method comprising illuminating the cell with the LACCprotein with a series of light pulses in which light periods are from0.1 ms to 100 ms), preferably from 1 ms to 50 ms, most preferablybetween 5 ms and 20 ms, and the periods of darkness were greater than 1ms, preferably greater than 10 ms, and most preferably greater than 20ms. The periods of darkness can be long if desired, and can be periodsof greater than seconds to minutes.

The methods of optically controlling cell properties of the presentinvention will result in the ability to probe causal function in intactneural circuits, with it becoming possible to examine the role ofparticular neurons in animal models of learning, emotion, motorcoordination, and sensory processing. This will enable the discovery ofdrugs capable of modulating whole-circuit function, essential for theaddressing of complex neurological and psychiatric diseases. For thefirst time, genetically-targeted neurons within animals will beaddressable by light on timescales appropriate for examining the neuralcode mediating the behavioral or circuit-dynamics function observed,whether normal or dysfunctional.

The millisecond scale control of electrical signals in neurons that ismade possible by the present invention creates the ability to controlsynaptic events. The ease of eliciting synaptic transmission allows LACCproteins of the present invention including ChR2 to be an ideal tool forthe temporally precise analysis of neural circuits.

Another aspect of the present invention is the use of light to create asynaptic event. A LACC protein of the present invention expressed withina neuron can be activated by light to create an electrical signal withinthe neuron. That electrical signal can propagate along the neuron to thesynapse where the signal can elicit a synaptic event. The synaptic eventcan be either an electrical or a chemical synaptic event. In a preferredembodiment the synaptic event releases a small molecule that canmodulate cellular function. In a preferred embodiment, the synapticevent comprises the release of a small-molecule neuromodulator such asnorepinephrine, serotonin, dopamine, acetylcholine, D-serine, orhistamine. The synaptic event can result in a synaptic transmissionbetween two neurons. The present invention thus provides opticallydriven communication between sets of neurons, and thereby providesoptically driven activity throughout a connected neural network.

A preferred embodiment for the use of light to create a synaptic eventcomprises expressing ChR2 in a first neuron and illuminating the neuronwith pulse of light to activate ChR2 to create a spike which causes asynaptic event. The use of light to create synaptic events can becarried out in-vitro or in-vivo.

Another preferred embodiment for the use of light to create a synapticevent comprises expressing ChR2 in a first neuron and that in synapticcontact with a second neuron, and illuminating the first neuron so as tocreate a spike, wherein the spike results in a synaptic event at thesynapse between the first and second neurons, and the second neuron iseither excited or inhibited due to the signal from the first neuron,thus resulting in a synaptic transmission between the first and secondneurons.

Another preferred embodiment for the use of light to create a synapticevent comprises using a vector that is targeted to deliver the LACCprotein of the present invention to specific neurons, specific subsetsof neurons, or specific neuronal subtypes. The targeted delivery can bea specific spatial targeting into different spatial areas of thespecimen, and/or the targeted delivery can be to chemical targeting tomolecularly defined classes or subclasses of neurons.

Targeted Delivery of LACC

An aspect of the invention is a method for targeted delivery oflight-activated cation-channel proteins to specific cells comprising;contacting said cells with a vector comprising a nucleic acid sequencecomprising a LACC protein and a cell specific promoter; wherein saidspecific cells express said LACC protein.

A preferred embodiment of a method for targeted delivery oflight-activated cation-channel proteins to specific cells comprises amethod wherein the vector comprises a nucleic acid sequence that codesfor Chop2 or ChR2 and a cell specific promoter. Preferred cell specificpromoters are the promoters for somatostatin, parvalbumin, GABAα6, L7,and calbindin. Other cell specific promoters are promoters for kinasessuch as PKC, PKA, and CaMKII; promoters for other ligand receptors suchas NMDAR1, NMDAR2B, GluR2; promoters for ion channels including calciumchannels, potassium channels, chloride channels, and sodium channels;and promoters for other markers that label classical mature and dividingcell types, such as calretinin, nestin, and beta3-tubulin.

A preferred embodiment of a method for targeted delivery oflight-activated cation-channel proteins to specific cells comprises amethod wherein the vector comprises a lentivirus or a retrovirus.

An aspect of the invention is a method for targeted delivery oflight-activated cation-channel proteins to specific cells comprising;contacting said cells with a vector comprising a nucleic acid sequencecomprising a LACC protein wherein said vector selectively targetsspecific cells. Preferred vectors are lentiviruses and retrovirus.

Methods of Treatment

Another aspect of the invention is a method for treating a subjectcomprising delivering a vector comprising a LACC protein to excitablecells within the subject and illuminating said cells with pulses oflight.

A preferred embodiment of a method for treating a subject comprisesperforming human therapeutic functions in which the function of cells isrescued or controlled by the genetic addition of LACC proteins such asChR2, accompanied by the use of physically delivered pulses of light,preferably blue light. Delivering a LACC protein such as ChR2 in humanpatients via viral vectors can enable control of excitable cells by bluelight, either from a wearable optical device (for chronic stimulation)or at a fixed optical station (for more occasional stimulation). Forexample, peripheral neurons like cutaneous pain suppressing nerves,virally transduced to express ChR2, allow light stimulation to activatedorsal column-medial lemniscus neurons in order to suppress painful Cfiber responses. It has been shown that modified herpes viruses can beused to deliver ion channels to pain-pathway neurons; which can be usedherein with ChR2 for the targeting of the channel to pain-pathwayneurons and for reduction of the perception of pain. Similarly, patientswho have rod or cone loss (such as in retinitis pigmentosa or maculardegeneration) can be virally transduced to express a LACC protein suchas ChR2 in retinal ganglion cells, which restores the transduction oflight in pathways mediating visual perception. For instance, it has beenshown that long-term expression of a microbial-type rhodopsin,channelrhodopsin-2 (ChR2), can be achieved in rodent inner retinalneurons in vivo using delivery by an adeno-associated viral vector. Itwas demonstrated that expression of ChR2 in surviving inner retinalneurons of a mouse with photoreceptor degeneration can restore theability of the retina to encode light signals and transmit the lightsignals to the visual cortex (Bi et al. Neuron 50, 23-33 (2006)). Thus,the strategy based on the expression of ChR2 is suitable for retinaldegenerative diseases. In another example, ChR2-expressing T lymphocytesthat attack cells bearing self-antigens can be induced to undergoapoptosis where illumination induces significant Ca+2 influx; this canbe done ex vivo on blood that passes through an optical illuminationdevice.

In one embodiment, the optical device used to excite LACCprotein-expressing cells in patients is a light-emitting diode (LED).The LED can be in the millimeter or nanometer scale in size. Onenon-limiting example includes SML0805-B1K-TR LEDtronics. The LED can bebattery-powered or remotely power by RF by methods that are known in theart. The LED can also be couple to a 68-microHenry surface-mount ferritecore inductor such as CF1008-682K from Gowana Electronics, NY. In oneembodiment, the wearable optical device can be non-invasively activatedin a non-wireless fashion.

The methods and compositions provided herein can provide a beneficialeffect for depression patients. Preferably depression patients aretreated by delivering and exciting a LACC protein such as ChR2 to theanterior and/or subgenual cingulate cortex and to anterior limb ofinternal capsule of human patients by the methods described herein.

The methods and compositions provided herein can also provide abeneficial effect for chronic pain patients. Preferably chronic painpatients are treated by delivering and exciting a LACC protein such asChR2 to the anterior and/or dorsal cingulate cortex of human patients bythe methods described herein.

Similarly, the methods and compositions provided herein can provide abeneficial effect for obesity patients. Preferably obesity patients aretreated by delivering and exciting a LACC protein such as ChR2 to theventromedial nucleus of the thalamus of human patients by the methodsdescribed herein.

Similarly, the methods and compositions provided herein can provide abeneficial effect for obsessive compulsive (OCD) patients. PreferablyOCD patients are treated by delivering and exciting a LACC protein suchas ChR2 to the anterior limb of internal capsule subthalamic nuclei ofthe thalamus of human patients by the methods described herein.

Similarly, the methods and compositions provided herein can provide abeneficial effect for addiction patients. Preferably addiction patientsare treated by delivering and exciting a LACC protein such as ChR2 tothe nucleus accumbens and septum of human patients by the methodsdescribed herein.

Similarly, the methods and compositions provided herein can provide abeneficial effect for Alzheimer's patients. Preferably Alzheimer'spatients are treated by delivering and exciting a LACC protein such asChR2 to hippocampus of human patients by the methods described herein.

Similarly, the methods and compositions provided herein can provide abeneficial effect for Parkinson's patients. Preferably Parkinson'spatients are treated by delivering and exciting a LACC protein such asChR2 to the subthalamic nuclei and/or globus pallidus of human patientsby the methods described herein.

Another route for human therapy using a LACC protein such as ChR2 is tocreate a LACC protein-expressing secretory cell for implantation inpatients (for example, nanoencapsulated to avoid immune responses) inwhich secretion is stimulated in the cells by the use of physicallydelivered pulses of light. For example, ChR2-expressing neuroendocrinecells that release thyroid hormones (such as T4, TRH, and others) can beimplanted subcutaneously to allow for controlled peptide release overtimescales from months to years. Sequences of flashes of light allowcontrolled release of such neuroendocrine substances, allowingmodulation of stress, reproduction, metabolism, and sleep. Similarly,LACC protein-expressing pancreatic islet cells can be made to releaseinsulin when stimulated with light; implanted cells can enable controlof diabetes symptoms on a minute-to-minute timescale without need forpump implantation or other invasive therapy.

In one embodiment, LACC protein-expressing cells are encapsulated priorto implantation into patients. The cells can be macroencapsulated ornanoencapsulated. Examples of capsules include but are not limited tosemipermeable membranes, hollow fibers, beads and planar diffusiondevices. For example, encapsulation of dopamine-secreting cells in asemipermeable membrane has been examined to avoid rejection of implantedcells by the immune system (Emerich et al. Neurosci Biobehav Rev 16,437-447). The selectively permeable nature of the polymer membranepermits bidirectional access of low molecular weight compounds,including the inward diffusion of glucose, oxygen and other vitalnutrients, and the outward diffusion of dopamine. The membrane restrictsthe passage of elements of the host immune system, thereby preventinghost rejection of the encapsulated cells. For example, it has been shownthat encapsulated PC12 cells, a catecholaminergic cell line derived fromrat pheochromocytoma, can be implanted into the striatum of monkeys andadult guinea pigs (Date et al. Cell Transplant 9, 705-709, Aebischer etal. Exp Neurol. 111, 269-275). In addition, it has been shown thatxenotransplantation of islets or insulin producing cells encapsulatedwithin hollow fibers, macrobeads, or planar diffusion devices canreverse hyperglycemia in mice and rats (Tatarkiewicz et al.Transplantation 67(5) 665-671).

In another embodiment, LACC protein-expressing cells are generated inthe capsule prior to transplantation. For instance, ES cells can be: i)differentiated into dopamine producing neurons, ii) transfected with anucleic acid containing LACC as described above, and iii) grown in acapsule, prior to implantation into patients. It has been shown thatdopaminergic neurons can be induced from ES cells from mouse enclosed inhollow fibers using conditioning medium from PA6 cells, the stromalcells derived from skull bone marrow (Yamazoe et al. Biomaterials 27(2006) 4871-4880). In these studies β-tubulin type III positive cellsand tyrosine hydroxylase positive cells were efficiently derived inhollow fibers after 16 days in culture, and dopamine release wasobserved when the hollow fibers containing cells were exposed to 56 mMKCl for 15 min to induce dopamine release through depolarization of theneurons.

In another embodiment, differentiated LACC protein-expressing stem cellscapable of secreting dopamine would be implanted, directly into thebrain of a patient, and then drive their activation using light.Dopamine-secreting cells can be transfected or infected as describedherein with a LACC protein such as ChR2, before or after thedifferentiation step, and then these cells can be implanted into thebrain of the patients. The LACC protein-expressing stem cells are thenactivated by an optical device such as a light-emitting diode or a laserwith an optical fiber attached to the end. Since the cells are lightresponsive, they will release dopamine even deep within tissue, simplyby remotely being stimulated with light.

In another embodiment, LACC protein-expressing secretory cells areimplanted into a tissue or an organ of a patient. The secretory cell istransfected or infected as described herein with a LACC protein such asChR2, and then these cells are implanted into the tissue or organ of thepatient. The LACC protein-expressing secretory cells are then induced tosecrete chemicals by an optical device such as a light-emitting diode ora laser with an optical fiber attached to the end. FIG. 10 illustratesone embodiment of the methods described herein, wherein cells expressingChR2 (by transfection or infection) allow release of substances to becontrolled by pulses of light.

Examples of tissues or organs that can be implanted with LACCprotein-expressing secretory cells include, but are not limited toepithelium, connective tissue, connective tissue, nervous tissue, heart,lungs, brain, eye, stomach, spleen, pancreas, kidneys, liver,intestines, skin, uterus, and bladder.

Examples of chemicals that can be secreted into the tissue or organ of apatient by the methods described herein include but are not limited toinsulin, growth hormone, fast neurotransmitter, dopamine, cytokines,chemokines, hormones and hormone antagonists, pituitary hormones andtheir hypothalamic releasing factors, thyroid and antithyroid drugs,estrogens and progestins, androgens, adrenocorticotropic hormone;adrenocortical steroids and their synthetic analogs; inhibitors of thesynthesis and actions of adrenocortical hormones, insulin, oralhypoglycemic agents, and the pharmacology of the endocrine pancreas,agents affecting calcification and bone turnover: calcium, phosphate,parathyroid hormone, vitamin D, calcitonin, vitamins such aswater-soluble vitamins, vitamin B complex, ascorbic acid, fat-solublevitamins, vitamins A, K, and E, growth factors, muscarinic receptoragonists and antagonists; anticholinesterase agents; agents acting atthe neuromuscular junction and/or autonomic ganglia; catecholamines,sympathomimetic drugs, and adrenergic receptor agonists or antagonists;and 5-hydroxytryptamine (5-HT, serotonin) receptor agonists andantagonists.

In one embodiment, LACC protein-expressing secretory cells are implantedinto the skin of a diabetic or patient. The LACC protein-expressingsecretory cells are then induced to secrete insulin by an optical devicesuch as a light-emitting diode or a laser with an optical fiber attachedto the end.

A LACC protein such as ChR2 allows for temporal, noninvasive control ofrelease of substances from excitable cells. It should be apparent thatthe electrical activity, Ca+2, and secretion of different chemicals canaffect literally thousands of cellular processes. Thus, it is possiblethat the general arena of applications of a LACC protein such as ChR2 invivo may enable control over many biological disease processes in avariety of systems.

Controlling Organism or Cells

Another aspect of the present invention is a method of controlling thebehavior of an organism comprising delivering a vector comprising a LACCprotein to excitable cells within the organism; and illuminating saidcells with pulses of light so as to control the behavior of theorganism.

Another aspect of the invention involves the use of the method describedherein to control the fate of cells. The techniques described herein areused to modulate activities of cells that are modulated by electricalactivity. The methods can be used to modulate the survival, replication,differentiation, and/or death of cells. Channelrhodopsin is used todrive any one of these processes, depending on the precise pattern ofstimulation used to drive activation of channelrhodopsin, which wouldthen result in a specific pattern of downstream signal transduction anda specific cellular fate response. Thus, targeting Channelrhodopsin tospecific cells, then exposing them to particular light patterns is usedto enhance the survival of, drive differentiation or replication of, orhasten the death of, the cells expressing the light-activated channel.This modulation of cellular processes is preferably used in stem cells,where particular patterns of activity may drive the differentiation ofstem cells (including human embryonic stem cells), drive the replicationof stem cells, or drive the death of the stem cells (in the case whereexcessive replication is desired to cease). If the target cells aretumor or cancer cells, then targeting channelrhodopsin to those cellspermits the use of specific and appropriate patterns of light to killthe tumor/cancer cells. Other suitable target cells include varioussecretory or organ cells or their precursors, cardiac or other musclecells, or glial cells in the brain. In each of these cases, it may bedesirable to control the replication, differentiation, and death ofthese cells precisely. Channelrhodopsin may be useful for controllingthese things in vitro, in vivo in experimental animals, or in vivo inhumans (before or after transplantation into the body).

Drug Screening

An aspect of the invention is a method for screening for drugscomprising expressing a LACC protein in a group of cells; exposing saidgroup of cells to a compound; illuminating said groups of cells withlight; and monitoring the electrical response of cells within the groupof cells. The electrical response of cells can be monitoredelectrically, optically, or by other means. The electrical responses ofthe cells can be seen and monitored using chemical or voltage-sensitivedyes, or dyes that undergo oxidation or reduction. These changes can bemonitored in a microscope, with optical sensors or with a camera usingfilm, CCD arrays, and other methods known by persons of skill in theart.

A preferred embodiment of a method of drug screening is high-throughputscreening for drugs affecting the ionic and signaling function of cellssuch as central and peripheral neurons, heart muscle, pancreatic isletcells, neuroendocrine cells in pituitary and kidney, stem cells, cancercells, and others.

A preferred embodiment of a method of drug screening comprises rapidlymodulating voltage in electrically excitable cells capable of fastresponses (such as neurons and heart muscle, or cell lines derivedthereof), to screen for drugs that block or activate voltage-gated ionchannels, alter excitatory or inhibitory synaptic transmission (play arole in epilepsy and pain), and alter muscular contraction. Since manychannels and receptors activate rapidly, and then desensitize orinactivate, the ability to control membrane voltage with brief pulses oflight greatly enhances the ability to develop drugs that target certainchannel, synapse, or muscle disease phenotypes. For example, familialhemiplegic migraine patients have point mutations in a calcium channel,reducing the calcium influx, for a given brief depolarization. Themethods of the present invention will allow for screening drugs thatmodulate the calcium response (measured using a calcium dye) to a givenstereotyped depolarization, in a cell line expressing the mutant calciumchannel and ChR2, to discover ways of improving channel function in thispatient population.

There are many cells that do not always rapidly change voltage, butnevertheless have significant functions in the body downstream ofelectrically-driven Ca+2 influx. For example, pancreatic islet cells andneuroendocrine cells (e.g., those of the thyroid, pituitary, and adrenalglands) release hormones in a Ca+2-dependent way, via the fusion ofdense core vesicles. LACC proteins of the present invention such asChR2, which not only causes cell depolarization but is capable ofpassing Ca+2 ions through its channel pore, can be used to activate therelease of peptides or hormones from such cells (or derived cell lines),aiding in the screening of drugs that enhance or suppress hormonerelease. Such discoveries can aid in the discovery of pharmacologicalmethods of treating problems of impaired growth, development,metabolism, stress, and reproduction.

Finally, the LACC proteins of the present invention including. ChR2 canbe used to screen for drugs that alter slow signal transductionprocesses in cells, revealing treatments for chronic disease processesranging from cancer to depression. In particular, immune cells respondto ongoing patterns of Ca+2 influx with a variety of long-lastingchanges, which can lead to strengthening or weakening of immuneresponses, or even possibly autoimmune symptoms. Cancer cells alsoexpress ion channels, and may respond to electrical activity withaltered proliferation. Neural stem cells respond to depolarization andCa+2 influx by differentiating into neurons; activating stem cellsexpressing LACC proteins with light can then enhance survival andincorporation into mature tissues, a step in generation of functionalbrain and other tissue.

In a preferred embodiment of a method of drug screening, the stem cellline expressing a LACC protein of the present invention such as ChR2 canbe used to optically control tissue repair, and illumination of thesestem cells with light results in CREB phosphorylation, a critical stepin enhancing their transformation into neurons.

Another preferred embodiment is a method of drug screening comprisesmaking a human embryonic stem (ES) cell line with ChR2 under a stem-cellspecific promoter to build cells which differentiate under opticalcontrol. Expression of ChR2 in such cells allows discovery of drugswhich alter the activity-dependent progress of cellular life and deathamidst their environments.

Using the methods of the present invention, cells can be forced tochange developmental fate via remote control, and a drug screening toolwhere this is possible allows the discovery of new molecules thatmanipulate such long term effects of cell proliferation,differentiation, and apoptosis. In all of these scenarios, specific cellpopulations will be light-addressable on rapid timescales, allowing thescreening of many aspects of cellular function.

These techniques are suitable for screening for drugs that modulate ionchannel function (blocking or facilitating ion channel function—eitherby blocking the pore, affecting the gating, or affecting the openchannel). FIG. 11 illustrates one example of the methods for screeningdrugs. First, the baseline electrical activity of the ion channel ismeasured by flashing the light and observing the depolarization. Thefluorescence of the cells will return to its original value after thelight is off. Then a drug is applied, for example, by pipetting in thedrug (or flowing in through a microfluidic channel). Finally, the lightis re-flashed and the electrical activity of the ion channel ismeasured. This technique can be used in combination with the whole-cellpatch clamp technique, wherein an electrode is placed up to a cell,suction is applied to break into the cell, and to record the activity ofthe cell expressing an ion channel before and after the application of adrug. Examples of drugs that can be screen according to the methodsdescribed herein include, but are not limited to, antidepressants,antipsychotics, calcium antagonist, antiepilecticts, as well as drugs totreat OCD, Alzheimer's, Parkinson, obesity, addiction, chronic pain,muscle and heart disorders.

One embodiment of a method for drug screening is as follows:

-   -   1) express a light activated channel protein, preferably        Channelrhodopsin, in a cell line;    -   2) express an ion channel of interest (“channel n”) in the same        cell line;    -   3) label the cells with a voltage sensitive dye (or other        suitable indicator, such as those described below);    -   4) expose said cells to light, and record the fluorescence of        the voltage sensitive dye;    -   5) expose said cells to a candidate compound that possibly        modulates the function of channel n;    -   6) expose said cells to light a second time, and record the        fluorescence of the voltage sensitive dye.        Preferably, the modulation of the light activated channel        protein is related to the modulation of the channel n. For        example, activation of the light activated channel protein        causes an activation of channel n. This co-relation in activity        can be direct or indirect. The co-relation can be via a signal        transduction pathway. The co-relation in activity can be        directly proportional to each other or inversely proportional to        each other.

If the fluorescence is greater during step (6) than step (4), presumablythe candidate drug facilitates channel function. If the fluorescence issmaller during step (6) than step (4), presumably the candidate drugdiminishes channel function. If the fluorescence is equal in steps (4)and (6) (allowing for any bleaching of the dye), then the drug does notaffect channel function. In this way, drugs that affect channel functioncan be detected rapidly. FIG. 11 illustrates one example in which thedrug is a negative modulator of channel function

After preparation of the cell line in steps (1) and (2), the cell linewould suffice for the screening of many (perhaps millions of) drugs,which modulate channel n. Steps (4), (5), and (6) can take place in arobotic device that moves a 384-well plate into the focus of an opticalbeam. The wells of the plate could all contain the same cell line,facilitating the screening of drugs that affect a particular channel(“high throughput target screening,” see below), or each well couldcontain cells of a different cell line, facilitating the screening ofone drug against many different channels (“screening against sideeffects,” see below).

A voltage sensitive dye is preferred due its fast kinetics, but otherdyes (e.g., a calcium-sensitive dye in the case that channel n is acalcium channel) could also serve to indicate whether channel functionis modulated by the drug. Genetically encoded indicators of voltage orcalcium may also be useful for reading out the activity of the cell(e.g., FLASH, GCaMP2, cameleon, etc.). In this case, these indicatorswould be stably expressed in the cell line as well. Other methods ofreading out whether the drug had an effect are also useful—e.g.,immunostaining for the phosphorylation of a site that is phosphorylatedduring or after periods of ion channel activity.

For drug screening, it is preferred to have an optical imaging devicecontaining 1) a light source (light-emitting diode (LED), lamp, laser)for illuminating the cell expressing Channelrhodopsin and driving achange in cell voltage, 2) a light source for illuminating a dye orindicator, possibly the same light source as used for driving thevoltage change, and 3) a switch for alternating between the two lightsources or a beamsplitter for simultaneous non-interfering delivery ofboth kinds of light. The fluorescence of the dye or indicator istypically measured by a sensor (CCD camera, PMT, or photodiode). Thiskind of device is useful for ion channel drug screening, as describedabove. The device itself may also consist of a robotic arm for moving aplate (e.g., a 384-well plate) through the arena where the light sourcesand sensor are present. This kind of combined light source/imagingdevice has also diagnostic applications. For example, taking cells froma patient, expressing Channelrhodopsin in them, and then exposing themto light, could be used to detect patient-specific ion channel syndromesin biopsy samples or in cells of the circulatory system.

The methods described herein are preferably performed withchannelrhodopsin, but any suitable light-activated cation channel can beused.

Yet another aspect of the invention is methods for high-throughputtarget screening. In one embodiment, high-throughput target screeningcomprises a plate reader that moves wells containing cells of interestinto the optical stimulation/readout area, and then performs the methodsdescribed above and illustrated in FIG. 11. Then the machine moves theplate to enable the next well to be flashed, in turn. Examples of wellplates include 96-, 384, 1526-well plates. In one embodiment, thehigh-throughput target screening methods of the invention can analyze upto about 2,000 drugs per day per setup. In another embodiment, thehigh-throughput target screening methods of the invention can analyze upto about 2,500,000 drugs per year per 5 setups.

In another embodiment, high-throughput target screening comprises achip-based system. A chip-based system can enable sequential rather thanparallel testing of compounds on ion channels by high-speed scanning ofa single cell across a laminar stream of solution environments createdin the microfluidic chips. The scanning of the microfluidic chip,causing a cell to sample the discrete zones of drug solutions, iscontrolled by a computer controlled motorized scan stage. Themicrofluidic chips, for example, can have eight-channel, 16-channel,48-channels or more for high-throughput analysis.

In another embodiment, high-throughput target screening comprises a cellsorting device that includes an array of discrete locations forcapturing cells traveling along a fluid flow. The discrete locations canbe arranged in a defined pattern across a surface such that the discretesites are also addressable and contain discrete zones of drug solution.Examples of surfaces that may be used for creating arrays of cells indiscrete sites include, but are not limited to, cellulose, celluloseacetate, nitrocellulose, glass, quartz or other crystalline substratessuch as gallium arsenide, silicones, metals, semiconductors, variousplastics and plastic copolymers, cyclo-olefin polymers, variousmembranes and gels, microspheres, beads and paramagnetic orsupramagnetic microparticles.

Yet another aspect of the invention is methods for screening againstside effects. This method allows for screening for drugs thatselectively affect one ion channel, but do not affect other ionchannels. This screening against individual ion channels allows forscreening to predict potential side effects.

As an example of screening for side effects: many channels are expresseddifferentially in the heart vs. the brain. By screening for drugs thatdifferentially bind to channels in the brain, but not the heart, it ispossible to find neuropsychiatric drugs that don't affect heartfunction. Especially important for heart function are calcium channels,HERG channels, other potassium channels, and other ion channels thataffect the rhythmicity or amplitude of the heartbeat.

Prosthetic Devices

Another aspect of the invention is the use of the methods andcompositions described herein in prosthetic devices. Blindness,deafness, and other sensory deficits affect millions of peopleworldwide, severely impacting their quality of life. Channelrhodopsin,targeted to somatic cells in the human patient, opens up a new class ofsensory prostheses. For example, some forms of blindness destroyphotosensor function, but leave signal processing in downstream neuronsintact. In such diseases, such as macular degeneration or retinitispigmentosa, targeting Channelrhodopsin to retinal ganglion cells (forexample, by injecting viruses expressing channelrhodopsin into theretinal cell layers inside the eye) could enable restoration of visualfunction. In such patients treated with channelrhodopsin targeted toretinal ganglion cells, the retinal ganglion cells would themselvesbecome photosensitive, enabling vision with resolution comparable to thenative eye, and preferably not requiring invasive technology beyond thatpoint. Channelrhodopsin is sufficiently sensitive to detect sunlight(power ˜1 kW/m^2). Alternatively, the Channelrhodopsin can be targetedto amacrine cells or bipolar cells to enable vision. It is also possiblethat expressing channelrhodopsin in a retinal cell, accompanied with aprojection device that would amplify the ambient light, would enablevision indoors or in low-light conditions.

In another embodiment, in many age- and experience-related forms ofdeafness, hair cells are lost, but downstream neurons are intact.Expressing Channelrhodopsin in spiral ganglion cells (i.e., eight nerveneurons) would enable activation of these cells with light. Cochlearimplants currently stimulate all the cells of the cochlea with a singleelectrode or at most a few electrodes, and do not attempt torecapitulate any of the spatial distribution of sensory afferents in thecochlea. By inserting a device containing 1) a microphone to detectsound, 2) a microprocessor to analyze the frequency components of thesound and convert them to LED signals, and 3) multiple LEDs for emittinglight in a spatially patterned fashion, into the cochlea, atonotopically-mapped stimulator could be created, which would drivedifferent frequencies of sound perception simply by targeting the lightto appropriate cells.

In yet another embodiment, central nervous system neurons in a human areinfected with virus expressing Channelrhodopsin (or otherwise come toexpress Channelrhodopsin), these neurons become capable of responding tolight. This gene therapy approach allows optical stimulation of neuronaltargets in the brain. If the targeted neurons are in sensory cortex,this opens up the possibility of a new kind of cortical sensoryprosthesis. If the targeted neurons are in the frontal cortex or otherparts of the brain, it is in possible that these light-sensitive neuronspermit modulation of emotion or cognition. If the targeted neurons werein the spinal cord, it is possible that neurons that inhibit painfulstimuli could be driven by light. In general, this gene therapy approachopens up a new kind of generalized prosthetic, in which light isconverted into neural activity, in defined parts of the nervous system.

One embodiment of a prosthetic device, is for implanted cells that areengineered to secrete compounds and to respond to light, and also forhunting for neural-circuit level targets in the intact animal brain, itmay be very useful to have an implantable or head-mounted LED, or othersmall light source. Such a light source could be implanted under theskin, under the skull, deep within the brain, or deep within anotherorgan of interest, in which Channelrhodopsin-expressing cells are alsolocated (either exogenously introduced, or endogenously located andtargeted with a virus). This device could be used for stimulating ChR2in cells located directly adjacent to the light source. For the exampleof the cochlear implant, a strip of LEDs, each individuallycontrollable, could be useful. For the example of the cortical implant,a 2-dimensional array of LEDs could be useful. For the example of aninsulin-secreting cell located under the skin, a wearable blue LED on abracelet, powered by a battery, could be useful.

In some embodiments, for medical applications, the LEDs are remotelypowered. A remotely-powered LED could be made by combining an LED in aclosed-loop series circuit with an inductor. This would allowradiofrequency (RF) energy or rapidly changing magnetic fields (e.g.,delivered by a transcranial magnetic resonance (TMS) coil) totemporarily power-up the inductor, and thus the connected LED, allowinglocal delivery of light, even deep in a brain structure. Such a devicecould be implanted under the skin, under the skull, deep within thebrain, or deep within another organ, of interest in whichChannelrhodopsin-expressing cells are also located (either exogenouslyintroduced, or endogenously located and targeted with a virus). Then adevice that can remotely deliver RF or magnetic energy could be placednearby, or worn on the patient, for activating the implanted device.

Biochemical Modifications

Another aspect includes biochemical modifications of light activatedchannels, such as Channelrhodopsin. Such modifications are typicallyperformed to target Channelrhodopsin to different parts of a cell.Fusing channelrhodopsin to a targeting sequence of DNA, so that theresultant protein contains both channelrhodopsin and the targetingpeptide, could be used to send Channelrhodopsin to the presynapticterminal, the postsynaptic terminal, the nucleus, or other intracellularcompartments. Such targeting sequences include PDZ domains, glutamateand GABA receptor C-terminal sequences, ion channel c-terminalsequences, presynaptic scaffolding targeting sequences, and othertargeting sequences. These versions of Channelrhodopsin could be used totrigger specific intracellular signaling events, including thoseimportant for neuroprotection, memory, or other enduring signalingfunctions.

In a combinatorial fashion, these reagents could complement the otherapplications of Channelrhodopsin. For example: these reagents could beuseful for drug screening (e.g., finding drugs that modulate thefunction of a channel in a particular subcellular compartment). Thesereagents could also be useful for prosthetic devices (e.g., drivingactivity on the dendrites of a neuron, to more closely mimic naturalsynaptic activity).

The methods and devices are described herein with LEDs, but a smalllaser, or a fiber optic cable that carries light from an external source(a xenon or mercury lamp) can also be used. Preferred light sources usedto illuminate the Channelrhodopsin-expressing cells have the followingproperties:

stimulation times tunable from 0-25 ms, or even longer

brightnesses tunable in the range 0-10 mW/mm^2, or even higher

wavelengths in the range 440-490 nm, or broader depending on theidentity of the light-activated channel

Articles of Manufacture

In another aspect of the invention, articles of manufacture containingthe compositions described herein (e.g. a nucleic acid comprising anLACC sequence or a LACC protein-expressing cell) are provided. Thearticle of manufacture comprises a container and a label. Suitablecontainers include, for example, bottles, vials, syringes, plates, andtest tubes. The containers may be formed from a variety of materialssuch as glass or plastic. Suitable packaging and additional articles foruse (e.g., measuring cup for liquid preparations, foil wrapping tominimize exposure to air, dispensers, and the like) are known in the artand may be included in the article of manufacture.

In one embodiment, the container is a 96-, 384-, and 1526-well platecontaining different cells of interest in each well, for example,prefrozen in a protective medium (e.g. medium containing 20% DMSO). Theplates can be shipped in dry ice and store upon receipt at −80 degreesCelsius for short term storages. Alternatively, the plates are stored inliquid nitrogen for long term storage. This plate fit into aplate-reader/motorized stage device as the ones described above or aregular plate reader known in the art.

In one embodiment, the container holds a composition that is effectivefor treating a disease condition and may have a sterile access port (forexample the container may be an intravenous solution bag or a vialhaving a stopper pierceable by a hypodermic injection needle). Theactive agent in the composition is the compositions of the inventiondescribed herein (e.g. a nucleic acid comprising an LACC sequence or aLACC protein-expressing cell). The article of manufacture can furthercomprise, within the same or a separate container, another agent such asa therapeutic agent that is co-administered with the compositions of theinvention and optionally a pharmaceutically acceptable buffer, such asphosphate-buffered saline, Ringer's solution and dextrose solution.

The label on, or associated with, the container indicates instructionsto use the composition, for instance, the container may indicated thatthe composition is used for treating a disease condition of choice orfor screening compounds or screening compound's side effects. Thecontainers described herein may further include other materialsdesirable from a commercial and user standpoint, including otherbuffers, diluents, filters, needles, syringes, and package inserts withinstructions for use.

It is understood that the examples described herein in no way serve tolimit the true scope of this invention, but rather are presented forillustrative purposes. All references and sequences of accession numberscited herein are incorporated by reference in their entirety.

EXAMPLES

Plasmid Constructs.

The ChR2-YFP gene was constructed by in-frame fusing EYFP (Clontech) tothe C-terminus of the first 310 amino acid residues of ChR2 (GeneBankaccession no. AF461397) via a NotI site. The lentiviral vector pLECYTwas generated by PCR amplifying ChR2-YFP with primers:

5′-GGCAGCGCTGCCACCATGGATTATGGAGGCGCCCTGAGT-3′ (SEQ ID NO: 4) and5′-GGCACTAGTCTATTACTTGTACAGCTCGTC-3′ (SEQ ID NO: 5) and ligating intopLET (gift from Eric Wexler and Theo Palmer, Stanford University) viathe AfeI and SpeI restriction sites. The plasmid was amplified and thenpurified using Qiagen MaxiPrep kits (Qiagen).

Viral Production.

VSVg pseudotyped lentiviruses were produced by triple-transfection of293FT cells (Invitrogen) with pLECYT, pMD.G, and pCMVdeltaR8.7 (giftsfrom Eric Wexler and Theo Palmer) using Lipofectamine 2000. Thelentiviral production protocol is same as previously described⁴² exceptfor the use of Lipofectamine 2000 instead of calcium phosphateprecipitation. After harvest, viruses were concentrated by centrifugingin a SW28 rotor (Beckmann Coulter) at 20,000 rpm for 2 h at 4° C. Theconcentrated viral titer is determined via FACS sorting to be between5×10^8 and 1×10^9 IU/mL.

Hippocampal Cell Culture.

Hippocampi of postnatal day 0 (P0) Sprague-Dawley rats (Charles River)were removed and treated with papain (20 U/ml) for 45 mM at 37° C. Thedigestion was stopped with 10 ml of MEM/Earle's salts withoutL-glutamine along with 20 mM glucose, Serum Extender (1:1000), and 10%heat-inactivated fetal bovine serum containing 25 mg of bovine serumalbumin (BSA) and 25 mg of trypsin inhibitor. The tissue was trituratedin a small volume of this solution with a fire-polished Pasteur pipette,and 100,000 cells in 1 ml plated per coverslip in 24-well plates. Glasscoverslips (prewashed overnight in HCl followed by several 100% EtOHwashes and flame sterilization) were coated overnight at 37° C. with1:50 Matrigel (Collaborative Biomedical Products, Bedford, Mass.). Cellswere plated in (Life Technologies). One-half of the medium was replacedwith culture medium the next day, giving a final serum concentration of1.75%. No all-trans-retinal was added to the culture medium or recordingmedium for any of the experiments here described. However, B27 containssmall amounts of retinal derivatives like retinyl acetate, which mayhave assisted with ChR2 function. Additional supplementation withall-trans-retinal, or its precursors, may assist in the application ofChR2 to the studying of intact circuits.

Viral Infection.

Hippocampal cultures were infected on d.i.v. 7 using 5-fold serialdilutions of lentivirus (˜1×10^6 IU/mL) Viral dilutions were added tohippocampal cultures seeded on coverslips in 24 well plates and thenincubated at 37° C. for 7 d before recording using patch-clampingtechnique.

Confocal Imaging.

Images were acquired on a Leica TCS-SP2 LSM confocal microscope using a63× water-immersion lens. Cells expressing ChR2-YFP were imaged liveusing YFP microscope settings, in Tyrode's solution containing (in mM):NaCl, 125; KCl, 2; CaCl₂, 3; MgCl2, 1; glucose, 30; and HEPES, 25 (pH7.3 with NaOH). Propidium iodide (PI; Molecular Probes) staining wascarried out on live cells by adding 5 μg/mL PI to the culture medium for5 minutes at 37° C., washing twice with Tyrode solution, and thencounting fluorescent and nonfluorescent cells immediately. Coverslipswere then fixed for 5 minutes in PBS plus fresh 4% paraformaldehyde,permeabilized for 2 minutes with PBS plus 0.1% Triton X-100, and thenimmersed for 5 minutes in 5 μg/mL PI for observation of any pyknoticnuclei. At least eight different fields were examined per coverslip.

Electrophysiology and Optical Methods.

Cultured hippocampal neurons were recorded at approximately d.i.v. 14, 7days after infection. Neurons were recorded using the whole-cell patchclamp technique, using Axon Multiclamp 700B (Axon Instruments, Inc.)amplifiers on an Olympus IX71 inverted scope equipped with a 20×objective lens. Borosilicate glass (Warner) pipette resistances were onaverage 4 MΩ, range 3-8 MΩ. Access resistance was 10-30Ω and wasmonitored throughout the recording. Intracellular solution consisted of97 potassium gluconate, 38 KCl, 0.35 EGTA, 20 HEPES, 4 magnesium ATP,0.35 sodium GTP, 6 NaCl, and 7 phosphocreatine (pH 7.25 with KOH).Neurons were perfused in Tyrode's solution, described above. Allexperiments were performed at room temperature (22-24° C.). For allexperiments except for the synaptic transmission data shown in FIGS. 4band 4c , we patched fluorescent cells immersed in Tyrode solutioncontaining 5 μM NBQX and 20 μM gabazine to block synaptic transmission.

Photocurrents were measured while holding neurons in voltage clamp at−65 mV. Recovery from inactivation was measured by measuringphotocurrents while illuminating neurons with pairs of pulses lasting500 ms each, separated by periods of darkness lasting 1-10 seconds.

Spiking was measured while injecting current to keep the voltage of thecell at approximately—65 mV (holding current ranging from 0 pA to 200pA). For the synaptic transmission experiments, we patchednonfluorescent neurons near ChR2-expressing neurons, immersed in Tyrodesolution containing either 20 μM gabazine to isolate just the excitatorypostsynaptic response, or in 5 μM NBQX to isolate just the inhibitorypostsynaptic response. To confirm whether the evoked potentials wereindeed synaptically driven, after synaptic stimulation we blocked allpostsynaptic receptors with solution containing both 20 μM gabazine and5 μM NBQX, and rephotostimulated.

pClamp 9 software (Axon Instruments) was used to record all data and tooperate the MultiClamp 700B amplifier, and a Sutter DG-4 ultrafastoptical switch with 300W xenon lamp (Sutter Instruments) was used todeliver the light pulses for ChR2 activation. A standard Endow GFPexcitation filter (excitation filter HQ470/40x, dichroic Q495LP; Chroma)was used for delivering blue light for ChR2 activation, in the bandwidth450-490 nm. YFP was visualized with a standard YFP filter (excitationHQ500/20x, dichroic Q515LP, emission HQ535/30m; Chroma). Through a 20×objective lens, power density of the blue light was 8-12 mW/mm2, asmeasured with a silicon power meter (Newport). Pulse sequences weresynthesized by custom software written in MATLAB (MathWorks) and thenexported through pClamp 9 via a Digidata input/output board (Axon),attached to a PC.

Poisson trains were 8 seconds long, with Poisson parameter 2=100 or 200ms. For Poisson trains, a 10 ms-minimum refractory period was imposedbetween light pulses, for biophysical realism.

Membrane resistance was measured in voltage clamp mode with 20 mV pulseslasting 75 ms, and repeated every 3 seconds. Spike rates due to directcurrent injection were measured with pulses of 300 pA current lasting0.5 seconds.

Data Analysis.

Data was analyzed automatically using Clampfit (Axon) and customsoftware written in MATLAB. Spikes were extracted by looking forcrossings of the voltage above a threshold (typically 60 mV aboveresting potential), and latencies were measured from the onset of thelight pulse to the spike peak. Extraneous spikes were measured as thenumber of extra spikes after a single light pulse, plus any spikesoccurring later than 30 ms after the onset of a light pulse.

Jitter was calculated as the standard deviation of spike latencies,measured either throughout the spike train (when gauging reliabilitythroughout a train), or for the same spike across different trains (whengauging reliability across trials, or across neurons). For all jitteranalyses, light pulses that did not elicit a spike in a particularneuron were ignored for the analysis of jitter of that neuron. For theacross-neurons jitter analysis shown in FIG. 2h , light pulses that didnot elicit spikes in all 7 neurons were ignored (leaving 31/59 lightpulses for the λ=100 stimulus, and 30/46 light pulses for the λ=200stimulus).

Example 1

To obtain stable and reliable ChR2 expression for coupling light toneuronal depolarization, we constructed lentiviruses containing aChR2-YFP fusion protein for genomic modification of neurons. Infectionof cultured rat CA3/CA1 neurons led to appropriately membrane-localizedand well-tolerated expression of ChR2 for days to weeks after infection(FIG. 1a ). There was no evidence of toxicity due to the expressedprotein, even at high fusion protein expression levels. Whole-cellvoltage-clamp recording of neurons showed that conventional GFPillumination in the bandwidth 450-490 nm (300W xenon lamp (Sutter DG-4),via Chroma excitation filter HQ470/40x) induced depolarizing currentswith fast rise rates—reaching a maximal rise rate of 160+111 pA/mswithin 2.3+1.1 ms after the onset of the light pulse (mean±standarddeviation reported throughout, n=18; FIG. 1b , left). Mean whole-cellinward currents were large, 496 pA±336 pA at peak and 193 pA±177 pA atsteady-state (FIG. 1b , middle). In control experiments, light-evokedresponses were never seen in cells expressing YFP alone (data notshown). Consistent with the known excitation spectrum of ChR2²⁰,illumination of ChR2-expressing neurons with YFP-spectrum light in thebandwidth 490-510 nm (300 W xenon lamp filtered with Chroma excitationfilter HQ500/20x) resulted in smaller currents than those evoked withthe GFP filters (FIG. 1b , right). Despite the inactivation of ChR2 withsustained light exposure (FIG. 1b and ref.²⁰), we observed rapidrecovery of peak ChR2 photocurrents in neurons (FIG. 1c ; τ=5.1+1.4seconds; recovery trajectory fit with Levenberg-Marquardt algorithm;n=9). This rapid recovery is consistent with the well-known stability ofthe Schiff base (the lysine in transmembrane helix 7, which bindsretinal) in microbial-type rhodopsins, and the ability of retinal tore-isomerize to the all-trans ground state in a dark reaction withoutthe need for other enzymes. In addition to the short-term recovery ofpeak photocurrents shown above, light-evoked current amplitudes werealso stable over long timescales, remaining unchanged in patch-clampedneurons throughout an hour of pulsed light exposure (data not shown),confirming at the functional level the lack of toxicity suggested by theconfocal images (FIG. 1a ). Thus ChR2 can mediate rapid and sustainablephotocurrents of large amplitude, without detectable adverse sideeffects.

Example 2

We examined whether ChR2 could drive actual depolarization of neuronsheld in current-clamp mode, with the same steady illumination protocolwe used for eliciting ChR2-induced currents (FIG. 1d , left). Early inan epoch of steady illumination, single neuronal spikes were rapidly andreliably elicited (8.0+1.9 ms latency to spike peak, n=10; FIG. 1d ,right), consistent with the fast rise times of ChR2 currents describedabove. However, for these cells, at these specific conditions, anysubsequent spikes elicited during steady illumination were poorly timed(FIG. 1d , left). Thus, for this particular sample, steady illuminationwas not adequate for controlling the timing of ongoing spikes with ChR2,despite the reliability of the first spike. Earlier patch-clamp studiesusing somatic current injection showed that spike times were morereliable during periods of rapidly rising membrane potential than duringperiods of steady high-magnitude current injection. This is consistentwith our finding that steady illumination evoked a single reliably timedspike, followed by irregular spiking.

Example 3

We found that the single spike reliably elicited by steady illuminationhad extremely low temporal jitter from trial to trial (FIG. 1d , right;0.5+0.3 ms; n=10 neurons). This observation led us to devise apulsed-light paradigm, which takes advantage of the low jitter of thesingle reliable spike evoked at light pulse onset. But in order for sucha pulsed-light paradigm to work, the conductance and kinetics of ChR2would have to permit peak currents of sufficient amplitude, during lightpulses shorter than the desired interspike interval. Indeed, using fastoptical switching, we found that multiple pulses of light withinterspersed periods of darkness could elicit reliable and well-timedtrains of spikes (FIG. 1e ; shown for 25 Hz trains of four pulses). FIG.1e highlights the fact that longer light pulses evoke single spikes withgreater probability than short light pulses. The ability to easily alterlight pulse duration with fast optical switching suggests astraightforward method for eliciting spikes even in multiple neuronspossessing different ChR2 current densities, by simply increasing thelight pulse duration until single spikes are reliably obtained in allthe neurons being illuminated. Rapid modulation of light power wouldalso allow for this kind of control. In the experiments described here,we used light pulse durations of either 5, 10, or 15 ms (n=13 highexpressing neurons fired reliable spikes; n=5 low-expressing neuronsfired reliable subthreshold depolarizations). Thus, the nonlinear natureof neuronal spike production allow us to elicit spikes reliably, simplyby increasing the light energy delivered to the ChR2-expressing neuronuntil the resultant voltage deflection is above the threshold forspiking. Without the use of brief light pulses, however, it is possiblethat the fast kinetics of ChR2 would not serve as usefully in thecontrol of reliable spike induction: this highlights the need foroptical equipment to be matched to the bandwidth of the photostimulation reagent.

Example 4

The millisecond-scale control discovered herein raised the prospect ofgenerating arbitrarily defined, even naturalistic spike trains (such asPoisson trains, used commonly to model natural activity) in neurons byremote optical control. FIG. 2a shows spike trains in a hippocampalneuron in response to three deliveries of the same Poisson distributedseries of light pulses (here shown for a light pulse series 59 pulseslong, each lasting 10 ms; Poisson parameter λ=100 ms). Theseoptically-driven spike trains were quite consistent across repeateddeliveries of the same series of light pulses: on average, >95% of thelight pulses in a series elicited spikes during one trial if and only ifthey elicited spikes on a second trial, for both the λ=100 ms seriesshown in FIG. 2a , and a λ=200 ms series comprising 46 spikes (FIG. 2b ;n=7 neurons). Following the strategy of increasing light pulse durationto the point of reliable spiking, we used trains of light pulses lasting10 ms each for 4 of the 7 neurons, and trains of light pulses lasting 15ms for the other 3 (for the analyses of FIG. 2, all data were pooled).The trial-to-trial jitter was very small across repeated deliveries ofthe same Poisson series of light pulses (2.3+1.4 ms and 1.0+0.5 ms forλ=100 and λ=200 respectively; FIG. 2e ). Throughout extended pulseseries, the efficacy of eliciting spikes throughout the train wasmaintained (76% and 85% percent of light pulses successfully evokedspikes, respectively; FIG. 2d ). The latencies to spike after lightpulse onset were also consistent throughout the series of pulses(14.3+3.1 ms and 13.3+3.4 ms respectively; FIG. 2e ). Finally, spikejitter remained remarkably small throughout the train (3.9+1.4 ms and3.3+1.2 ms; FIG. 2e ). Hence, pulsed optical activation of ChR2 canelicit precise, repeatable spike trains in a single neuron, over time.

Example 5

Even across different neurons, activation of ChR2 by defined series oflight pulses could elicit the same spike train with strikingly highfidelity (shown for three hippocampal neurons in FIG. 2f ). Although theheterogeneity of individual neurons—for example, in their membranecapacitance (68.8+22.6 pF) and resistance (178.8+94.8 MΩ)—might beexpected to introduce significant variability in their integrativeelectrical properties, the strong nonlinearity inherent in the couplingof light to spiking overcame this variability. Indeed, different neuronsresponded in similar ways to a given light pulse series, with 80-90% ofthe light pulses in a train eliciting spikes in at least 4 of the 7 theneurons examined (FIG. 2g ). Moreover, spikes had very low temporaljitter when measured for the same light pulse series delivered todifferent neurons (3.4+1.0 ms and 3.4+1.2 ms for λ=100 and λ=200respectively; FIG. 2h ). Remarkably, this across-neuron jitter (FIG. 2h) was identical to the within-neuron jitter, measured throughout thelight pulse series (FIG. 2e ). Thus, heterogeneous populations ofneurons can be controlled in concert, with practically the sameprecision observed for the control of single neurons over time.

Example 6

Having established the ability of ChR2 to drive sustained naturalistictrains of spikes, we turned next to probing quantitatively the frequencyresponse of light-spike coupling. ChR2 enabled driving of sustainedspike trains from 5 to 30 Hz (FIG. 3a ; here tested with series oftwenty 10-ms long light pulses), as suggested by the Poisson train data(FIG. 2). For these particular cells under these particular conditions,it was easier to evoke more spikes at lower frequencies, than at higherfrequencies (FIG. 3b ; n=13 neurons). Light pulses delivered at 5 or 10Hz could elicit arbitrarily long spike trains (FIG. 3b ), with spikeprobability dropping off at higher frequencies of stimulation (20 Hzyielded 7.2+6.6 spikes, and 30 Hz yielded 4.0+6.3 spikes). For theseexperiments, the light pulse durations used were 5 ms (n=1), 10 ms(n=9), or ms (n=3) long (data from all n=13 cells were pooled for thepopulation analyses of FIG. 3). As expected from the observation thatlight pulses generally elicited single spikes (FIG. 1d and FIG. 2),almost no extraneous spikes occurred during the delivery of trains oflight pulses (FIG. 3c ). Even at higher frequencies, the temporal jitterof spike timing remained very low throughout the trains (<5 ms; FIG. 3d), and the latency to spike remained constant across frequencies (˜10 msthroughout; FIG. 3e ). Thus ChR2 can induce spiking across aphysiologically relevant range of firing frequencies, appropriate fordriving trains and bursts of spikes.

Example 7

It has been found that trial-to-trial variability in the subthresholddeflections evoked by repeated light pulses was quite small, withcoefficient of variation 0.06+0.03 (FIG. 4b ; n=5). ChR2 therefore canbe employed to drive reliably timed subthreshold depolarizations withprecisely determined amplitude.

Example 8

The high fidelity control of spiking mediated by ChR2 suggested that itwould be possible to optically drive activity throughout connectedneural networks, via synaptic transmission. Indeed, both excitatory(FIG. 4c ) and inhibitory (FIG. 4d ) synaptic events could be evoked inneurons receiving input from ChR2-expressing presynaptic neurons. Theseresults suggest that synaptic transmission can be controlled reliablywith ChR2.

Example 9

Extensive controls were carried out to test whether expression of ChR2in neurons perturbed their basal electrical properties, altered theirdynamic electrical properties in the absence of light, or jeopardizedtheir prospects for cellular survival. Lentiviral expression of ChR2 forat least one week did not alter neuronal membrane resistance (212+115MΩ2 for ChR2+ cells vs. 239.3+113 MΩ for ChR2− cells; FIG. 5a ; p>0.45;n=18 each) or resting potential (−60.6+9.0 mV for ChR2+ vs. −59.4+6.0 mVfor ChR2-; FIG. 5b ; p>0.60), measured in darkness. This suggests thatin neurons, ChR2 has little basal electrical activity, or even passiveshunting ability, in the absence of light. It also suggests thatexpression of ChR2 did not lead to impairment of general cell health, asindicated by electrical determination of membrane integrity. As anindependent measure of membrane integrity and cell health, we stainedlive cultured neurons with the membrane impermeant DNA-binding dyepropidium iodide (PI). ChR2+ expression did not affect the percentage oflive neurons that took up PI (1/56 ChR2+ neurons vs. 1/49 ChR2-neurons;p>0.9 by χ² test). Neither did we see any pyknotic nuclei, indicative ofapoptotic degeneration, in cells expressing ChR2 (data not shown). Wealso checked for alterations in the dynamic electrical properties ofneurons, measured in darkness. There was no difference in the voltagechange resulting from 100 pA of current injected in either thehyperpolarizing (−22.6+8.9 mV for ChR2+ vs. −24.5+8.7 mV for ChR2−;p>0.50) or depolarizing (+18.9+4.4 mV for ChR2+ vs. 18.7+5.2 mV forChR2−; p>0.90) direction, nor was there any difference in the number ofspikes evoked by a half-second+300 pA current injection (6.6+4.8 forChR2+ vs. 5.8+3.5 for ChR2-; FIG. 5c ; p>0.55). Thus, in the absence oflight, the presence of ChR2 does not alter cell health or ongoingelectrical activity, at the level of subthreshold changes in voltage orin spike output, either by shunting current through leaky channels or byaltering the voltage dependence of existing neuronal input-outputrelationships. These controls also suggest that there were nosignificant long-term plastic or homeostatic alterations in theelectrical properties of neurons expressing ChR2.

Example 10

In a test of whether ChR2 predisposes neurons to light-induced problemswith cellular health, we measured the electrical properties describedabove, after 24 hours in darkness post light-exposure to a typical pulseprotocol (1 sec of 20 Hz 15-ms light flashes, once per minute, for 10minutes). Exposure of neurons expressing ChR2 to light did not altertheir basal electrical properties relative to non-flashed neurons, withcells possessing normal membrane resistance (178+81 MΩ; FIG. 5a ;p>0.35; n=12) and resting potential (−59.7+7.0 mV; FIG. 5b ; p>0.75).Exposure to light also did not predispose neurons to cell death, asmeasured by live-cell PI uptake (2/75 ChR2+-neurons vs. 3/70ChR2-neurons; p>0.55 by χ² test). Neurons expressing ChR2 and exposed tolight flashes also had normal numbers of spikes elicited from somaticcurrent injection (6.1+3.9; FIG. 5c ; p>0.75). Thus, the membraneintegrity, cell health, and basal electrical properties were normal inneurons expressing ChR2 and exposed to light.

Example 11—Retroviruses for Insertion of ChR2

Moloney-type retroviruses selectively target dividing cells (Ory et al.,Proc Natl Acad Sci USA 93:11400 (1996)), such as stem cells. We haveconstructed retroviruses containing ChR2, either fused to a fluorescentprotein, or with an IRES to allow concomitant expression of a non-fusionfluorescent protein (FIG. 6). These viruses drive ChR2 under theCMV-Chicken beta actin promoter, and are VSV-G pseudotyped to permitefficacious infection of dividing mammalian cells. These retroviruseshave been generated by triply transfecting the plasmid containing ChR2with helper vectors into 293T cells. Alternatively, we have grown 293GPGcell lines in growth medium containing tetracycline, puromycin, and G418to select for cells sustainably carrying the retroviral packagingsystem. Tetracycline is used to suppress the expression of VSV-G, whichis toxic to 293GPG cells, during growth phase. After cells grow to 70%confluence, then tetracycline is removed to allow the expression ofVSV-G. At this point, the plasmid containing Chr2 and GFP (right) aretransfected into the 293GPG cells using Lipofectamine, and cultured293GPG cells are monitored for signs of fluorescence as gene expressionand retrovirus production begins. These cell lines are then frozen forfuture production of retrovirus. These viruses have been shown toefficaciously infect dividing cells, both in vitro (kidney cell lines,stem cells) and in vivo in rats (hippocampal stem cells, glia). Thisconstruct will be useful for targeting ChR2 to dividing cells ex vivo,for transplantation into humans for therapy, for the creation of celllines, and for the development of animals with selective populations oflabeled cells for studies of diseases of cellular activity, such asepilepsy, migraine, narcolepsy, and even autoimmune diseases.

The lentiviruses that we have generated contain tetracycline elementsthat allow control of the gene expression levels of ChR2, simply byaltering levels of exogenous drugs such as doxycycline. This method, orother methods that place ChR2 under the control of a drug-dependentpromoter, will enable control of the dosage of ChR2 in cells, allowing agiven amount of light to have different effects on electricalactivation, substance release, or cellular development. The lentivirusesthat we have generated contain tetracycline elements that allow controlof the gene expression levels of ChR2, simply by altering levels ofexogenous drugs such as doxycycline.

Example 12—Stem Cell Line

A clonal neural stem cell line that stably expresses ChR2-EYFP under thecontrol of the Ef1-alpha promoter was created by infecting cultured,nondifferentiated neural stem cells with a lentivirus containing thegene for ChR2. FIG. 7 shows a micrograph of cells from this neuralprogenitor cell (NPC) stem cell line. This cell line is appropriate forthe screening of drugs that affect the influence of electrical activityon neuronal genesis, development, or apoptosis. The stem cell line wehave already made is a correct step in realizing the goal of opticallycontrollable tissue repair, and illumination of these stem cells withlight results in CREB phosphorylation, a critical step in enhancingtheir transformation into neurons (FIG. 8). These homogeneous stem celllines can also be transplanted into the brain of adult animals, wherethey incorporate into hippocampal circuitry and can be studiedfunctionally after 2-6 weeks.

Example 13—Transgenic Zebrafish

Zebrafish (Danio rerio) embryos were acutely injected at the few-hundredcell stage with plasmid DNA containing ChR2 under promoters specific toparticular cell types in the zebrafish. A random subset of cells in thezebrafish then takes up the DNA, and keeps the plasmids during fishdevelopment; specific cells will then express the ChR2 in the plasmid.FIG. 9 shows micrographs of ChR2-EYFP selectively expressed in azebrafish trigeminal neuron (left) and in a zebrafish muscle cell(right). Briefly, larvae from 48 hours to 96 hours post fertilizationwere anesthetized in 0.02% tricaine in fish Ringer's solution andmounted in 1.2% agarose for imaging and photostimulation. Illuminationof the live fish expressing ChR2 in muscle with blue light for 0.5seconds caused rapid, phasic, single muscle contractions, which wasnever seen in zebrafish not containing ChR2. Neurons tolerated the ChR2expression well, and were able to be loaded with fluorescent calciumdyes (e.g., Oregon Green BAPTA-1, X-rhod-1), appropriate for themonitoring of neural activity downstream of a selectively activatedneuron expressing ChR2, using two-photon microscopy.

Example 14—Transgenic Fly

Flies (Drosophila melanogaster) have been generated that will expressChR2 under the UAS promoter, for use in the GAL4-UAS system that enablesflexible control of gene expression in flies. The UAS-ChR2 flies can becrossed with a variety of GAL4 lines of Drosophila, taking advantage ofthe large number of transgenic flies that have been created over thelast several years. We are crossing flies expressing UAS-ChR2 with fliescontaining GAL4 expressed exclusively in serotonergic and dopaminergicneurons. This will allow studies of the driving of motivated behaviorand the creation of finely tuned motor patterns.

Example 15—Transgenic Worm

Worms (C. elegans) have been caused to express ChR2 by the injection ofplasmids containing ChR2 under specific promoters into the syncytialgonad of the worm. Worms are also co-injected with a visible markergene, to allow visual inspection of the success of generating thetransgenic nematode. The gonad of the worm takes up the plasmid DNA andstores the DNA in large extrachromosomal arrays in eggs, passing theplasmid on to the worm's offspring. We have made C. elegans linestargeting ChR2 to the mechanosensory neuron AFD, the interneuron AIY,and also the serotonergic and dopaminergic neurons. These worms expressthe visible marker gene, indicating successful generation of stablelines. We are expressing ChR2 in C. elegans in serotonergic anddopaminergic neurons, important for the driving of motivated behaviorand the creation of finely tuned motor patterns.

Example 16—Transgenic Mouse

Mice can be made to express ChR2 transgenically under the control ofspecific promoters (by pronuclear injection of plasmids containing ChR2under a specific promoter), in specific loci (“knocking in” a gene intoan existing locus), using BAC transgenic technology (to mimic thenatural genetic environment of a gene), or by position effectvariegation techniques (a transgenic method allowing genes to randomlyexpress in tiny subsets of neurons). We are pursuing all of theseavenues. To rapidly demonstrate the power of this technology, we haveconstructed mice that will allow ChR2 to be expressed in small subsetsof neurons using position effect variegation. We have placed ChR2underneath the promoter for the gene Thy1, an immunoglobulin superfamilymember that is expressed by projection neurons in many parts of thenervous system (Gordon et al. Cell 50:445 (1987), Feng et al., Neuron28:41 (2000)). Previous transgenics expressing GFP under control of theThy1 promoter showed that many lines of the Thy1-GFP mice express GFP insmall subsets of neurons, due to random interactions of the Thy1promoter with local control elements (Feng et al., 2000). We haveinjected linearized plasmids containing Thy1-ChR2-EYFP into embryonicstem cells of mice, and these mice are currently being mated to produceoffspring expressing Thy1 in specific subsets of neurons in the nervoussystem. These mice will prove enormously powerful for the analysis ofthe function of previously unknown cell types in the brain, leading toan understanding of the causal function of specific neuronal types.

Example 17—Treatment of Photoreceptor Degeneration in Rodents with ChR2

The death of photoreceptor cells caused by retinal degenerative diseasesoften results in a complete loss of retinal responses to light Innerretinal neurons can be converted to photosensitive cells by deliveringchannelrhodopsin-2 (ChR2) using a lentivirus vector. The vectors can beconstructed as described in the examples above

Vector Injection—

Newborn (P1) rat (Sprague-Dawley and Long-Evans) and mouse (C57/BL andC3H/HeJ or rd1/rd1) pups can be anesthetized by chilling on ice. Adultmice (rd1/rd1) can be anesthetized by intraperitoneal injection of thecombination of katamine (100 mg/kg) and xylazine (10 mg/kg). Under adissecting microscope, an incision is made by scissors through theeyelid to expose the sclera. A small perforation is made in the scleraregion posterior to the lens with a needle and viral vector suspensionof 0.8-1.5 μl at the concentration of ˜13×10¹¹ genomic particles/ml canbe injected into intravitreal space through the hole with a Hamiltonsyringe with a 32-gauge blunt-ended needle. For each animal, usually,only one eye is injected with viral vectors carrying Chop2-GFP and theother eye is not injected or injected with viral vectors carrying GFPalone. After the injection, animals are kept on a 12/12 hr light/darkcycle. The light illumination of the room housing the animals measuredat the wavelength of 500 nm is typically 6.0×10¹⁴ photons cm⁻² s⁻¹.

The expression and functional properties of the ChR2 protein in thetransfected retinal neurons can be measured by methods known in the artincluding those described herein.

Visual-Evoked Potential Recordings—

Visual-evoked potential recordings are carried out in wild-type mice(C57BL/6 and 129/SV) at 4-6 months of age and in the rd1/rd1 mice at6-11 months of age and 2-6 months after the viral vector injection.After being anesthetized by intraperitoneal injection of the combinationof katamine (100 mg/kg) and acepromazine (0.8 mg/kg), animals aremounted in a stereotaxic apparatus. Body temperature is eithermaintained at 34° C. with a heating pat and a rectal probe orunregulated. Pupils are dilated by 1% atropine and 2.5%accu-phenylephrine. A small portion of the skull (˜1.5×1.5 mm) centeredabout 2.5 mm from the midline and 1 mm rostral to the lambdoid suture isdrilled and removed. Recordings are made from visual cortex (area V1) bya glass micropipette (resistance about 0.5 M after filled with 4 M NaCl)advanced 0.4 mm beneath the surface of the cortex at the contralateralside of the stimulated eye. The stimuli are 20 ms pluses at 0.5 Hz.Responses are amplified (1,000 to 10,000), band-pass filtered (0.3e100Hz), digitized (1 kHz), and are averaged between 30-250 trials.

Light Stimulation—

For visual evoked potential, light stimuli are generated by themonochromator and projected to the eyes through the optical fiber. Thelight intensity is attenuated by neutral density filters. The lightenergy is measured by a thin-type sensor (TQ82017) and an optical powermeter (Model: TQ8210) (Advantest, Tokyo, Japan).

Example 18—Treatment of Hyperglycemia in Mice by Transplantation ofMacronencapsulated ChR2 Transfected Islets

Macroencapsulated islets can reverse hyperglycemia in diabetic animalswhen transplanted i.p., s.c., or into the fat pad. Transplantation ofmacroencapsulated ChR2 transfected islets would provide a method forcontrol the release of insulin in a temporally precise manner. This canbe accomplished by transfecting islets with ChR2 and implanting thesecells into the skin of the animal and then driving their activationusing light.

Animals—

Male Swiss Webster nude mice (Taconic, Germantown, N.Y.) of 25-30 g canbe made diabetic with 250 mg/kg body weight (bw) i.p. injection of 4%streptozocin (STZ *) (Sigma, St. Louis, Mo.) dissolved in citratebuffer, pH 4.5. Only animals with blood glucose concentrations above 350mg/dl are then used as recipients. Sprague Dawley (SD) rats (Taconic) ofabout 250 g are used as donors. All animals would be kept underconventional conditions in acclimatized rooms with free access tostandard pelleted food and tap water. Nonfasting blood glucose levelsand body weight (bw) of recipients are measured on the day oftransplantation and then weekly for the next 7 weeks. Blood glucoseconcentrations can be measured with a One Touch II portable glucometer(Lifescan Inc., Milpitas, Calif.). Additional samples of plasma fromfreely fed and fasted normal mice and rats are collected to compare therelationship between plasma glucose and whole blood glucose in donor andrecipient strains. Plasma glucose values can be assessed by a GlucoseAnalyzer 2 (Beckman, Palo Alto, Calif.).

Islet isolation—

Rat islets are isolated technique known in the art. For example, 1-2mg/ml collagenase P (Boehringer Mannheim, Indianapolis, Ind.) solutionis injected into the pancreatic duct and the pancreas is digested for 19min at 37° C.; islets are then separated from the exocrine tissue usingdiscontinuous Histopaque-1077 (Sigma) density gradient centrifugation.Islets with diameters of 50-250 μm are hand picked, counted, andcultured in RPMI-1640 tissue culture medium with the standard glucoseconcentration of 200 mg/dl, supplemented with 10% fetal calf serum, 100U/ml penicillin, and 100 mg/ml streptomycin.

Islet can be transfected with ChR2 as described in the previousexamples. Expression and functional properties of ChR2 protein in thetransfected cells can be measured by methods known in the art includingthose described herein.

Macroencapsulation and transplantation—

Ported devices (Thera Cyte™) with an internal volume of 20 can be usedfor islet macroencapsulation. The devices are fabricated from membranelaminates composed of three layers: a cell-retentive membrane with anominal pore diameter of 0.45 μm laminated to an outer 5 μm porediameter polytetrafluoroethylene vascularizing membrane with an outerpolyester mesh providing support. The membrane laminates in arectangular shape is sealed on edges by ultrasonic welding. At one endof the device, polyethylene tubing is attached to provide access to thelumen. Aliquots of 1200 islets are suspended in a volume of 20 μl inEppendorf tubes and transferred through the port into the lumen of thedevice with a Hamilton syringe; the tubing is sealed with Silasticsilicon glue (Dow Corning Corp., Midland, Mich.). Transplantation can beperformed under Metofane anesthesia. When implanted s.c., a skinincision of about 2 cm is made on the back right side of the animal,followed by preparation of a s.c. pocket with gentle blunt dissection.The device is inserted into the pocket, and the incision was closed withwound clips.

The cells can be activated to release insulin with a light-emittingdiode or a laser with an optical fiber attached to the end or by themethods described herein.

Glucose Tolerance Tests—

Seven weeks after transplantation, IPGTTs are performed (glucose 2 g/kgbw, injected as a 10% solution). Before the glucose challenge, allanimals fast for about 16 hr. Samples from snipped tails for the bloodglucose are taken at 0, 15, 30, 60, 90 and 120 min after glucoseinjection.

Example 19—Generation of Dopaminergic ChR2 Transfected Neurons in HollowFibers

Effect of PA6 CM on ES Cell Differentiation—

ES cells are cultured using PA6 CM to induce neural differentiation. Toprepare PA6 conditioned medium (CM), confluent PA6 cells are washedthree times with phosphate-buffered saline with calcium and magnesiumions [PBS(+)] and then the respective media are replaced by G-MEMsupplemented with 5% KSR, 0.1 mM non-essential amino acids, 1 mMpyruvate and 0.1 mM 2-mercaptoethanol without/with heparin (10 or 100mg/ml). After 48 h, the supernatant is collected and filtered with a0.22 mm filter. CM collected using GMEM based media without, with 10mg/ml and with 100 mg/ml heparin are referred to as CM-OH, CM-10H andCM-100H, respectively.

ES cells are plated on polyornithine (Sigma)/fibronectin(Invitrogen)-coated glass plates at a density of 1250 cells/cm_2 inG-MEM supplemented with 5% KSR, 0.1 mM non-essential amino acids, 1 mMpyruvate and 0.1 mM 2-mercaptoethanol. After 5 h, CM-OH, CM-10H (finalconcentration of heparin is 3.3 mg/ml) or CM-100H (final concentrationof heparin is 33 mg/ml) is added to the culture plate to 13 volume ofthe culture medium and mixed well. All culture dishes are maintained at37° C. in air containing 5% CO2 for 16 days.

Differentiation of ES Cells in Hollow Fibers—

Hollow fibers, fabricated from semi-permeable polymer membranes andavailable in a variety of diameters and wall thickness, are bundled intoa housing and are used extensively in extracorporeal artificial organs,such as artificial kidney and artificial lung. Three different kinds ofhollow fibers can be used here. FB-150F fibers are used forHaemodialysis (NIPRO, Osaka, Japan) and are made of cellulose triacetatewith a 200 mm internal diameter and 15 mm thickness wall. Evafluxs 2Aand Evafluxs 5A fibers are used for plasmapheresis (Kuraray, Okayama,Japan) and are made of ethylene vinyl alcohol copolymer with a 175 mminternaldiameter and 40 mm thickness wall. Hollow fibers of FB-150F,Evafluxs 2A or Evafluxs 5A are referred to as HF-1, HF-2 or HF-3, andtheir molecular weight cutoff values are 55 kDa (95% cutoff), 144 kDa(90% cutoff) and 570 kDa (90% cutoff), respectively. ES cells aredissociated into single cells by treating them with 0.25% trypsin and 1mM EDTA in PBS. The cells are loaded into a 1 ml syringe at aconcentration of 1×10⁷ cells/ml and are gently injected into hollowfibers 9 cm in length. ES cell-loaded fibers are placed in 100 mm cellculture dishes and are maintained in G-MEM supplemented with 5% KSR, 0.1mM non-essential amino acids, 1 mM pyruvate, 0.1 mM 2-mercaptoethanoland 33% CM-10H. All culture dishes are kept in air containing 5% CO2 at37 1 C for 16 days.

ES cells can be transfected with ChR2 prior or after differentiation bythe methods described herein.

Cell growth of culture cells can be performed by methods known in theart. Differentiation of ES cells into dopaminergic neurons can beperformed by methods known in the art such as immunofluorescenceanalysis and RT-PCR. Dopamine levels can be determined by methods knowsin the art such as Reverse-phase HPLC.

The expression and functional properties of the ChR2 protein in thetransfected cells can be measured by methods known in the art includingthose described herein.

Generation of dopaminergic ChR2 transfected neurons in hollow fibers isa promising approach to perform cell therapy, for example, ofParkinson's disease. This method allows for the collection andencapsulation of dopaminergic ES progeny with minimal damage andprotection of implanted cells from the host immune system, which are twoof the obstacles encounter today. Furthermore, ChR2 protein expressionin the dopaminergic neurons allows for the control release of dopamineinto the brain according to the methods described herein. Whilepreferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby. Eliciting synaptic transmission demonstrates that ChR2is an ideal tool for the temporally precise analysis of neural circuits.

What is claimed is:
 1. A method of delivering a light activated cationchannel into a brain of a subject, the method comprising: a)administering directly to a portion of a brain in a subject a viralvector comprising a nucleotide sequence encoding a light activatedcation channel that comprises an amino acid sequence having at leastabout 95% amino acid sequence identity to the amino acid sequence setforth in SEQ ID NO: 1, wherein said nucleotide sequence is operablylinked to a promoter that provides for expression of the light activatedcation channel in a neuron in the portion of the brain, and wherein theportion of the brain is one or more of the anterior cingulate cortex,the subgenual anterior cingulate cortex, and the anterior limb ofinternal capsule; b) introducing a light source near the portion of thebrain; and c) activating the light activated cation channel in theneuron using light from the light source.
 2. The method of claim 1,wherein the step of activating the light activated cation channelprotein includes presenting a series of light pulses at a rate of atleast about 20 Hz.
 3. The method of claim 1, wherein the neuron is inthe anterior cingulate cortex of the brain.
 4. The method of claim 1,wherein the light activated cation channel protein is ChR2.
 5. Themethod of claim 1, wherein said activating comprises presenting a seriesof light pulses separated by less than about 50 milliseconds.
 6. Themethod of claim 1, wherein said promoter is a Thy1 promoter.
 7. Themethod of claim 1, wherein said light source is a light-emitting diode,a laser, or a fiber optic cable.
 8. The method of claim 1, wherein thelight has an intensity of from 0.1 mW/mm² to 50 mW/mm².
 9. The method ofclaim 1, wherein the light has a wavelength of from 440 nm to 590 nm.10. The method of claim 1, wherein the light has a wavelength of from440 nm to 490 nm.